US20220145261A1 - Pancreatic endocrine cells, method for producing same, and transdifferentiation agent - Google Patents
Pancreatic endocrine cells, method for producing same, and transdifferentiation agent Download PDFInfo
- Publication number
- US20220145261A1 US20220145261A1 US16/883,572 US202016883572A US2022145261A1 US 20220145261 A1 US20220145261 A1 US 20220145261A1 US 202016883572 A US202016883572 A US 202016883572A US 2022145261 A1 US2022145261 A1 US 2022145261A1
- Authority
- US
- United States
- Prior art keywords
- cells
- gene
- pancreatic endocrine
- vector
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000009996 pancreatic endocrine effect Effects 0.000 title claims abstract description 124
- 210000003890 endocrine cell Anatomy 0.000 title claims abstract description 120
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 70
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 267
- 210000001082 somatic cell Anatomy 0.000 claims abstract description 61
- 238000000034 method Methods 0.000 claims abstract description 58
- 101710096141 Neurogenin-3 Proteins 0.000 claims abstract description 20
- 241000131390 Glis Species 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims description 132
- 210000002950 fibroblast Anatomy 0.000 claims description 21
- 101150111723 PDX1 gene Proteins 0.000 claims description 19
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 claims description 16
- 101150088814 GLIS1 gene Proteins 0.000 claims description 15
- 101100121956 Homo sapiens GLIS1 gene Proteins 0.000 claims description 15
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 9
- 210000000577 adipose tissue Anatomy 0.000 claims description 3
- 210000002919 epithelial cell Anatomy 0.000 claims description 3
- 210000003494 hepatocyte Anatomy 0.000 claims description 3
- 210000004698 lymphocyte Anatomy 0.000 claims description 2
- 210000002540 macrophage Anatomy 0.000 claims description 2
- 210000000663 muscle cell Anatomy 0.000 claims description 2
- 210000001178 neural stem cell Anatomy 0.000 claims description 2
- 101100121962 Homo sapiens GLIS3 gene Proteins 0.000 claims 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims 1
- 210000002570 interstitial cell Anatomy 0.000 claims 1
- 239000000243 solution Substances 0.000 description 162
- 239000013598 vector Substances 0.000 description 150
- 239000000047 product Substances 0.000 description 86
- 230000003612 virological effect Effects 0.000 description 85
- 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 75
- 238000012360 testing method Methods 0.000 description 69
- 108090001061 Insulin Proteins 0.000 description 43
- 239000005090 green fluorescent protein Substances 0.000 description 38
- 229940125396 insulin Drugs 0.000 description 38
- 102000004877 Insulin Human genes 0.000 description 37
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 27
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 27
- 239000012228 culture supernatant Substances 0.000 description 25
- 239000003795 chemical substances by application Substances 0.000 description 24
- 241001430294 unidentified retrovirus Species 0.000 description 24
- 241000699666 Mus <mouse, genus> Species 0.000 description 21
- 230000014509 gene expression Effects 0.000 description 19
- 239000008103 glucose Substances 0.000 description 19
- 239000007758 minimum essential medium Substances 0.000 description 19
- 238000004458 analytical method Methods 0.000 description 16
- 239000013612 plasmid Substances 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 14
- 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 13
- 101000857270 Homo sapiens Zinc finger protein GLIS1 Proteins 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 13
- 101710183548 Pyridoxal 5'-phosphate synthase subunit PdxS Proteins 0.000 description 13
- 238000012258 culturing Methods 0.000 description 13
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 12
- 102100025883 Zinc finger protein GLIS1 Human genes 0.000 description 12
- 102100038553 Neurogenin-3 Human genes 0.000 description 11
- 108020004999 messenger RNA Proteins 0.000 description 11
- 101000857276 Homo sapiens Zinc finger protein GLIS3 Proteins 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 102100025879 Zinc finger protein GLIS3 Human genes 0.000 description 9
- 238000010367 cloning Methods 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 238000003753 real-time PCR Methods 0.000 description 9
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 8
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 8
- 230000003914 insulin secretion Effects 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 238000004806 packaging method and process Methods 0.000 description 8
- 229960004666 glucagon Drugs 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 229960000553 somatostatin Drugs 0.000 description 7
- 239000013603 viral vector Substances 0.000 description 7
- 102000051325 Glucagon Human genes 0.000 description 6
- 108060003199 Glucagon Proteins 0.000 description 6
- 102000005157 Somatostatin Human genes 0.000 description 6
- 108010056088 Somatostatin Proteins 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 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 6
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 6
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 5
- 210000004153 islets of langerhan Anatomy 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 230000001172 regenerating effect Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 101150112014 Gapdh gene Proteins 0.000 description 4
- 241000713869 Moloney murine leukemia virus Species 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 208000005017 glioblastoma Diseases 0.000 description 4
- 238000012744 immunostaining Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 210000000496 pancreas Anatomy 0.000 description 4
- 102200082402 rs751610198 Human genes 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000010195 expression analysis Methods 0.000 description 3
- 108010027225 gag-pol Fusion Proteins Proteins 0.000 description 3
- 210000005260 human cell Anatomy 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000002325 somatostatin-secreting cell Anatomy 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- 102100027723 Endogenous retrovirus group K member 6 Rec protein Human genes 0.000 description 2
- 101710121417 Envelope glycoprotein Proteins 0.000 description 2
- 101100484378 Fowlpox virus (strain NVSL) FPV055 gene Proteins 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 101000857273 Homo sapiens Zinc finger protein GLIS2 Proteins 0.000 description 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000015031 pancreas development Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 238000010079 rubber tapping Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-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
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 241000710189 Aphthovirus Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 101150029662 E1 gene Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 101710186643 Insulin-2 Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100121957 Mus musculus Glis1 gene Proteins 0.000 description 1
- 101100121960 Mus musculus Glis2 gene Proteins 0.000 description 1
- 101100121963 Mus musculus Glis3 gene Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 241000711975 Vesicular stomatitis virus Species 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 102100025884 Zinc finger protein GLIS2 Human genes 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 102000051956 human GLIS1 Human genes 0.000 description 1
- 102000056492 human GLIS2 Human genes 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 238000012528 insulin ELISA Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229910052754 neon Inorganic materials 0.000 description 1
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/14—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from hepatocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/25—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from renal cells, from cells of the urinary tract
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/30—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cancer cells, e.g. reversion of tumour cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/06—Uses of viruses as vector in vitro
Definitions
- the present invention relates to a method for producing pancreatic endocrine cells from somatic cells, pancreatic endocrine cells produced by the method, and a transdifferentiation agent that transdifferentiates somatic cells to pancreatic endocrine cells.
- Pancreatic endocrine cells have been expected to be used as, for example, a material for regenerative therapies for diabetes or a material used for screening of diabetes drugs.
- regenerative therapies for example, it has been expected that ⁇ cells, which are one of the pancreatic endocrine cells and produce insulin, are administered to type I diabetes patients who are insulin-deficient.
- ES cells embryonic stem cells
- iPS cells induced pluripotent stem cells
- the method has the following problems. Firstly, the method is complicated because culturing environments are needed to be properly adjusted by, for example, adding various inhibitors involved in development or differentiation to a cell culture medium. Secondly, the method may be unreproducible. Thirdly, the method is problematic in terms of efficiency because other cells than the ⁇ cells are also produced. Finally, the method takes at least 21 days to 30 days to produce the ⁇ cells, that is, the ⁇ cells are not capable of being produced in a short period of time.
- ES cells embryonic stem cells
- iPS cells induced pluripotent stem cells
- pancreatic endocrine cells the method being simple, easily reproduced, excellent in production efficiency, and capable of producing the pancreatic endocrine cells in a short period of time.
- GLIS1 GMS family zinc finger 1
- GLIS3 GLIS family zinc finger 3
- Ngn3 Neurogenin3
- PTL 1 Japanese Patent Application Laid-Open (JP-A) No. 2013-519371.
- the present invention aims to solve the above existing problems and achieve the following object. That is, the present invention has an object to provide a method for producing pancreatic endocrine cells, the method being simple, easily reproduced, excellent in production efficiency, and capable of producing the pancreatic endocrine cells in a short period of time; pancreatic endocrine cells produced by the method; and a transdifferentiation agent that transdifferentiates somatic cells to pancreatic endocrine cells.
- a method for producing pancreatic endocrine cells including
- a transdifferentiation agent including:
- transdifferentiation agent is configured to transdifferentiate somatic cells into pancreatic endocrine cells.
- the present invention can provide a method for producing pancreatic endocrine cells, the method being simple, easily reproduced, excellent in production efficiency, and capable of producing the pancreatic endocrine cells in a short period of time; pancreatic endocrine cells produced by the method; and a transdifferentiation agent that transdifferentiates somatic cells to pancreatic endocrine cells.
- FIG. 1 is a graph illustrating the measurement results of the number of DsRed2-positive insulin producing cells 12 days after viral infection in Test Examples 1.
- FIG. 2A is an image illustrating the analysis results of expression of insulin and somatostatin in Test Examples 2.
- FIG. 2B is an image illustrating the analysis results of expression of insulin and glucagon in Test Examples 2.
- FIG. 2C is an image illustrating the analysis results of expression of insulin and Pdx1 in Test Examples 2.
- FIG. 3A is a graph illustrating the results in dMEFs in Test Example 3.
- FIG. 3B is a graph illustrating the results in human iPS (253G13-6)-derived fibroblasts in Test Example 3.
- FIG. 3C is a graph illustrating the results in human HepG2 in Test Example 3.
- FIG. 3D is a graph illustrating the results in mouse NIH-3T3 in Test Example 3.
- FIG. 3E is a graph illustrating the results in human embryonic fibroblasts in Test Example 3.
- FIG. 3F is a graph illustrating the results in human neonatal fibroblasts in Test Example 3.
- FIG. 3G is a graph illustrating the results in human HEK293 in Test Example 3.
- FIG. 4A is a graph-1 illustrating the results in Test Example 4.
- FIG. 4B is a graph-2 illustrating the results in Test Example 4.
- FIG. 4C is a graph-3 illustrating the results in Test Example 4.
- FIG. 4D is a graph-4 illustrating the results in Test Example 4.
- FIG. 5A is a graph illustrating the results in human T98G glioblastoma in Test Example 5.
- FIG. 5B is a graph illustrating the results in human mesenchymal stem cells in Test Example 5.
- FIG. 6A is an image illustrating a pancreatic islet isolated from a mouse pancreas in Test Example 6.
- FIG. 6B is an image illustrating dMEFs infected with a retrovirus using a G1NP solution as a viral solution in Test Example 6.
- FIG. 7 is a graph illustrating the results of a glucose-responsive insulin secretion test in Test Example 7.
- FIG. 8 is a graph illustrating the results of a glucose-responsive insulin secretion test in Test Example 8.
- FIG. 9 is a graph illustrating the results in Test Example 9.
- Pancreatic endocrine cells of the present invention are capable of being produced by a method for producing pancreatic endocrine cells of the present invention.
- pancreatic endocrine cells of the present invention will now be described in conjunction with the method for producing pancreatic endocrine cells of the present invention.
- the method for producing pancreatic endocrine cells of the present invention includes at least an introduction step; and, if necessary, further includes other steps.
- the introduction step is a step of introducing one or more genes of a GLIS family or one or more gene products thereof and an Ngn3 gene or one or more gene products thereof into somatic cells.
- the gene products refer to mRNAs transcribed from genes or proteins translated from the mRNAs.
- the genes or one or more gene products thereof to be introduced into the somatic cells in the introduction step include at least one or more genes of a GLIS family or one or more gene products thereof and an Ngn3 gene or one or more gene products thereof; and, if necessary, further include other genes or one or more gene products thereof.
- a source of the one or more genes of the GLIS family is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include human and mouse.
- the one or more genes of the GLIS family are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include GLIS1, GLIS2, and GLIS3. These may be used alone or in combination. Among the one or more genes of the GLIS family, GLIS1 and GLIS3 are preferable, and GLIS1 is more preferable from the viewpoint of excellent production efficiency of the pancreatic endocrine cells.
- Sequence information of the one or more genes of the GLIS family is available from known databases.
- sequence information is available from NCBI under Accession numbers of NM_147193 (human GLIS1), NM_147221 (mouse GLIS1), NM_032575 (human GLIS2), NM_031184 (mouse GLIS2), NM_152629 (human GLIS3), NM_175459, and NM_172636 (mouse GLIS3).
- a source of the Ngn3 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include human and mouse.
- Sequence information of the Ngn3 gene is available from known databases.
- sequence information is available from NCBI under Accession numbers of NM_009719 (mouse) and NM_020999 (human).
- genes or one or more gene products thereof are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they do not impair effects of the present invention.
- a Pdx1 gene or one or more gene products thereof are preferable.
- a source of the Pdx1 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include human and mouse.
- Sequence information of the Pdx1 gene is available from known databases.
- sequence information is available from NCBI under Accession numbers of NM_000209 (human) and NM_008814 (mouse).
- Each of sequences of the one or more genes of the GUS family or one or more gene products thereof, the Ngn3 gene or one or more gene products thereof, and the other genes or one or more gene products thereof may consist of a region to be translated into a protein in the sequence of each of the genes, or may include other regions than the region to be translated into a protein.
- Each of the genes or one or more gene products thereof may have a mutation.
- mutations that do not change an amino acid sequence of a protein from each of the genes and mutations in which one or several (2 to 5) amino acids are deleted, substituted, inserted, or added in an amino acid sequence of a protein from each of the genes.
- a sequence homology to each of corresponding wild-type genes or one or more gene products thereof is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more in a base sequence of the region to be translated into a protein.
- the genes or one or more gene products thereof to be introduced into the somatic cells in the introduction step are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they include at least the one or more genes of the GUIS family or one or more gene products thereof and the Ngn3 gene or one or more gene products thereof.
- the genes or one or more gene products thereof preferably consist of (1) the one or more genes of the GUS family or one or more gene products thereof and the Ngn3 gene or one or more gene products thereof or (2) the one or more genes of the GUS family or one or more gene products thereof, the Ngn3 gene or one or more gene products thereof, and the Pdx1 gene or one or more gene products thereof, from the viewpoints of higher simplicity, easiness of reproduction, excellent production efficiency, and production of the pancreatic endocrine cells in a short period of time.
- Somatic Cells are not particularly limited and may be appropriately selected depending on the intended purpose.
- the somatic cells may be undifferentiated precursor cells or terminally differentiated mature cells.
- the somatic cells may be derived from ES cells or iPS cells.
- somatic cells include adipose tissue-derived interstitial (stem) cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, fibroblasts, hepatic cells, epithelial cells, renal cells, macrophages, lymphocytes, and muscle cells.
- stem adipose tissue-derived interstitial
- neural stem cells hematopoietic stem cells
- mesenchymal stem cells fibroblasts
- fibroblasts hepatic cells
- epithelial cells renal cells
- macrophages lymphocytes
- muscle cells fibroblasts, mesenchymal stem cells, hepatic cells, epithelial cells, and renal cells are preferable, and fibroblasts and mesenchymal stem cells are more preferable.
- a species of an individual from which the somatic cells are harvested is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include human and mouse.
- an individual from which the somatic cells are harvested is not particularly limited and may be appropriately selected depending on the intended purpose.
- the individual is preferably the individual oneself or other individuals having the same or substantially the same MHC type as that of the individual, in terms of a rejection reaction.
- substantially the same MHC type means, as used herein, that the MHC type is compatible to the extent that, when pancreatic endocrine cells derived from the somatic cells are transplanted into an individual, transplanted cells are capable of being engrafted with the use of, for example, an immunosuppressive agent.
- a time when the somatic cells are harvested from the individual is not particularly limited and may be appropriately selected depending on the intended purpose.
- a condition under which the somatic cells are cultured is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a culturing temperature of about 37° C. and a CO 2 concentration of from about 2% to about 5%.
- a medium in which the somatic cells are cultured is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include minimum essential media (hereinafter may be referred to as “MEM”), Dulbecco's modified Eagle media (hereinafter may be referred to as “DMEM”), RPMI1640 media, 199 media, and F12 media, all of which contain from 5% by mass to 20% by mass of serum.
- MEM minimum essential media
- DMEM Dulbecco's modified Eagle media
- RPMI1640 media RPMI1640 media
- 199 media 199 media
- F12 media all of which contain from 5% by mass to 20% by mass of serum.
- a method for introducing each of the genes or one or more gene products thereof into the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose.
- vectors, synthetic mRNA (messenger RNA), or recombinant proteins may be used.
- the vector is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include viral vectors and non-viral vectors.
- viral vectors include retroviral vectors and lentiviral vectors.
- non-viral vectors include plasmid vectors and episomal vectors.
- a method for introducing the vector into the somatic cells is not particularly limited and may be appropriately selected from known methods in the art.
- the methods described in, for example, WO 2007/69666; Cell, 126, 663-676 (2006); or Cell, 131, 861-872 (2007) may be used.
- the methods described in, for example, Science, 318, 1917-1920 (2007) the methods described in, for example, Science, 318, 1917-1920 (2007).
- viral particles obtained using packaging cells may be used.
- the packaging cells are cells into which viral structural protein-coding genes have been introduced.
- a recombinant viral vector into which a target gene has been incorporated is introduced into the cells, recombinant viral particles into which the target gene has been incorporated are produced.
- the packaging cells are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include packaging cells based on human kidney-derived HEK293 cells or mouse fibroblast-derived NIH3T3 cells; packaging cells Platinum-E (hereinafter may be referred to as “Plat-E cells”) which are capable of producing high titer viruses for a long period of time and in which viral structural proteins gag-pol and env are expressed under the control of MoMuLV (Moloney Murine Leukemia Virus) LTR (long terminal repeats); PLAT-A cells that are designed to express Amphotropic virus-derived envelope glycoproteins; and PLAT-GP cells that are designed to express vesicular stomatitis virus-derived envelope glycoproteins.
- MoMuLV Moloney Murine Leukemia Virus
- PLAT-A cells that are designed to express Amphotropic virus-derived envelope glycoproteins
- PLAT-GP cells that are designed to express vesicular stomatitis virus-derived envelope glycoprotein
- a method for introducing the viral vector into the packaging cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include lipofection methods, electroporation methods, and calcium phosphate methods.
- a method for infecting the somatic cells with the resultant viral particles is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include polybrene methods.
- the vector may include a marker gene for verifying whether each of the genes has been successfully introduced.
- the marker gene refers to a gene that allows for cell sorting or cell selection by introducing the marker gene into a cell.
- Specific examples of the marker gene include drug resistant genes, fluorescent protein genes, luminescent enzyme genes, and coloring enzyme genes. These may be used alone or in combination.
- drug resistant genes include neomycin resistant genes, tetracycline resistant genes, kanamycin resistant genes, zeocin resistant genes, and hygromycin resistant genes.
- fluorescent protein genes include green fluorescent protein (GFP) genes, yellow fluorescent protein (YFP) genes, and red fluorescent protein (RFP) genes.
- luminescent enzyme gene examples include luciferase genes.
- coloring enzyme genes include ⁇ galactosidase genes, ⁇ glucuronidase genes, and alkaline phosphatase genes.
- one gene may be incorporated into one vector, or two or more genes may be incorporated into one vector.
- the two or more genes may be expressed at the same time (hereinafter may be referred to as “co-expression”).
- a method for incorporating two or more genes into one vector is not particularly limited and may be appropriately selected depending on the intended purpose. However, the two or more genes are preferably incorporated via a linkage sequence.
- the linkage sequence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include gene sequences coding for a foot and mouth disease virus (Picornaviridae Aphthovirus)-derived 2A peptide and IRESs (internal ribosome entry sites).
- a method for introducing the mRNA into the somatic cells is not particularly limited and may be appropriately selected from known methods in the art.
- a method for introducing the recombinant protein into the somatic cells is not particularly limited and may be appropriately selected from known methods in the art.
- the number of times of introduction of each of the genes or one or more gene products thereof into the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose.
- each of the genes or one or more gene products thereof may be introduced once or two or more times.
- a time when each of the genes or one or more gene products thereof are introduced into the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose.
- the genes or one or more gene products thereof may be introduced at the same time or at different times.
- An amount of each of the genes or one or more gene products thereof to be introduced into the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose.
- the genes or one or more gene products thereof may be introduced in an equal amount or different amounts.
- the genes or one or more gene products thereof to be used may be the genes only, the gene products only, or both of the genes and the gene products.
- genes or one or more gene products thereof may be combined with a different gene or one or more gene products thereof.
- the combination is not particularly limited and may be appropriately selected depending on the intended purpose. The same combination or different combinations may be used for each of the genes or one or more gene products thereof.
- the other steps are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they do not impair effects of the present invention.
- Examples thereof include a genes or genes products thereof-introduced cells culturing step in which somatic cells, into which each of the genes or one or more gene products thereof has been introduced, are cultured.
- the genes or genes products thereof-introduced cells culturing step is a step of culturing somatic cells into which each of the genes or one or more gene products thereof has been introduced.
- a condition under which the genes or genes products thereof-introduced cells are cultured is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a culturing temperature of about 37° C. and a CO 2 concentration of from about 2% to about 5%.
- a medium used for culturing the genes or genes products thereof-introduced cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include MEM media, DMEM media, RPMI1640 media, 199 media, and F12 media, all of which contain from 5% by mass to 20% by mass of serum.
- a period of time of the genes or genes products thereof-introduced cells culturing step is not particularly limited and may be appropriately selected depending on the intended purpose.
- An exchange frequency of the medium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include every 2 days to 3 days.
- a method for verifying whether pancreatic endocrine cells are successfully produced by the method for producing pancreatic endocrine cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method in which expression of proteins to be expressed in the pancreatic endocrine cells is verified and a method in which expression of genes to be expressed in the pancreatic endocrine cells is verified.
- ⁇ cells of the pancreatic endocrine cells are produced is capable of being verified by the presence or absence of glucagon expression
- whether ⁇ cells of the pancreatic endocrine cells are produced is capable of being verified by the presence or absence of insulin expression
- ⁇ cells of the pancreatic endocrine cells are produced is capable of being verified by the presence or absence of somatostatin expression.
- the method in which expression of proteins is verified is not particularly limited and may be appropriately selected from known methods in the art. Examples thereof include immunostaining analyses.
- the method in which expression of genes is verified is not particularly limited and may be appropriately selected from known methods in the art. Examples thereof include quantitative PCR analyses.
- the pancreatic endocrine cells are capable of being produced from somatic cells through transdifferentiation. Therefore, the method is advantageous in that the pancreatic endocrine cells are capable of being produced without undergoing the iPS cell stage that have a risk of forming tumors.
- the transdifferentiation refers to direct transformation from a cell type to another cell type without undergoing the stem cell stage.
- the method for producing pancreatic endocrine cells of the present invention is simple and easily reproduced because a gene or one or more gene products thereof only have to be introduced into somatic cells, and at the same time the pancreatic endocrine cells are capable of being produced efficiently in a short period of time. Moreover, the method for producing pancreatic endocrine cells of the present invention is also advantageous in that the pancreatic endocrine cells are capable of being produced without using a special medium of which culturing environments are properly adjusted by, for example, adding inhibitors involved in development to the medium.
- the pancreatic endocrine cells may be ⁇ cells, ⁇ cells, ⁇ cells, or mixtures thereof. Among them, ⁇ cells are preferable in terms of regenerative therapies for diabetes patients.
- pancreatic endocrine cells of the present invention are suitably available as pancreatic endocrine cells used for screening of diabetes drugs.
- a transdifferentiation agent of the present invention is a transdifferentiation agent for transdifferentiating somatic cells into pancreatic endocrine cells.
- the transdifferentiation agent includes at least one or more genes of a GUIS family or one or more gene products thereof and an Ngn3 gene or one or more gene products thereof; and, if necessary, further includes other components.
- Somatic cells to be targeted by the transdifferentiation agent and preferable aspects thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- Pancreatic endocrine cells obtained using the transdifferentiation agent and preferable aspects thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- the one or more genes of the GUS family or one or more gene products thereof and preferable aspects thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- the one or more genes of the GUTS family or one or more gene products thereof may also have the same mutation as those described under the section entitled “Production method of pancreatic endocrine cells.”
- an aspect of the one or more genes of the GUIS family or one or more gene products thereof in the transdifferentiation agent is not particularly limited and may be appropriately selected depending on the intended purpose.
- the one or more genes of the GUS family or one or more gene products thereof may be incorporated into a vector, or may be a synthetic mRNA or a recombinant protein.
- the vector may be the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- the synthetic mRNA and the recombinant protein may be produced by any of known methods in the art.
- the Ngn3 gene or one or more gene products thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- the Ngn3 gene or one or more gene products thereof may also have the same mutation as those described under the section entitled “Production method of pancreatic endocrine cells.”
- Ngn3 gene or one or more gene products thereof in the transdifferentiation agent is not particularly limited and may be appropriately selected depending on the intended purpose.
- the Ngn3 gene or one or more gene products thereof may be incorporated into a vector, or may be a synthetic mRNA or a recombinant protein.
- the vector may be the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- the synthetic mRNA and the recombinant protein may be produced by any of known methods in the art.
- the other components are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they do not impair effects of the present invention.
- the other components preferably include a Pdx1 gene or one or more gene products thereof.
- the Pdx1 gene or one or more gene products thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- the Pdx1 gene or one or more gene products thereof may also have the same mutation those described under the section entitled “Production method of pancreatic endocrine cells.”
- An aspect of the Pdx1 gene or one or more gene products thereof in the transdifferentiation agent is not particularly limited and may be appropriately selected depending on the intended purpose.
- the Pdx1 gene or one or more gene products thereof may be incorporated into a vector, or may be a synthetic mRNA or a recombinant protein.
- the vector may be the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- the synthetic mRNA and the recombinant protein may be produced by any of known methods in the art.
- the genes or one or more gene products thereof in the transdifferentiation agent may be divided into separate containers or may be placed in a single container. Alternatively, any number of the genes or one or more gene products thereof may be placed in each container.
- An amount of each of the genes or one or more gene products thereof in the transdifferentiation agent is not particularly limited.
- the genes or one or more gene products thereof may be included in an equal amount or different amounts.
- the transdifferentiation agent may be suitably used as a component of a kit for producing pancreatic endocrine cells.
- the kit for producing pancreatic endocrine cells includes at least the transdifferentiation agent; and, if necessary, further includes other components.
- kit for producing pancreatic endocrine cells are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they do not impair effects of the present invention.
- examples thereof include packaging cells and media.
- the packaging cells and the media may be the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- Test Example 1 Production of Pancreatic Endocrine Cells from Mouse Fibroblasts-1
- Dual-labeled-mouse embryonic fibroblasts (hereinafter may be referred to as “dMEF”), which were one kind of somatic cells, were prepared in the following manner.
- mice in which pancreatic endocrine precursor cells are fluorescently labeled with GFP (mice expressing EGFP under the control of an Ngn3 gene promoter (Ngn3-eGFP)) were produced in the following manner.
- a construct in which a fusion protein gene of GFP and a nuclear localization signal (hereinafter may be referred to as “nls”) was ligated downstream of the Ngn3 gene promoter (5 kb) isolated from a BAC clone, was microinjected into about 400 fertilized eggs to thereby produce genetically modified mice in which pancreatic endocrine precursor cells are fluorescently labeled with GFP.
- nls nuclear localization signal
- mice in which pancreatic ⁇ cells are fluorescently labeled with DsRed2 (mice expressing DsRed2 under the control of a rat insulin promoter (Ins-DsR)) were produced in the following manner.
- a construct in which a DsRed2 gene was ligated downstream of the rat insulin promoter (800 bp), was microinjected into about 400 fertilized eggs to thereby produce genetically modified mice in which pancreatic ⁇ cells are fluorescently labeled with DsRed2.
- mice in which pancreatic endocrine precursor cells are fluorescently labeled with GFP were crossed with the genetically modified mice in which pancreatic ⁇ cells are fluorescently labeled with DsRed2, and then male and female offspring mice (heterozygous) were crossed with each other to generate dual-labeled genetically modified mice (Ngn3-eGFP/Ins-DsR) that were confirmed to be homozygous by genomic southern blotting. Two pairs (male and female) of the homozygous dual-labeled genetically modified mice were crossed.
- the remaining pellet was added with and suspended in 1 mL of a 0.25% trypsin-containing EDTA solution (available from Wako Pure Chemical Industries, Ltd., #201-16945, containing 0.25% DNase I), and then incubated in a water bath at 37° C. The water bath was stirred by hand every 10 min.
- the minced embryonic tissue corresponding to one animal was well-suspended in 5 mL of DMEM (containing 10% FBS) in a 15 mL tube, transferred into 5 mL of DMEM in a 10 cm cell culture dish, and then incubated within an incubator with 5% CO 2 at 37° C.
- DMEM phosphate-buffered saline
- PBS phosphate-buffered saline
- One milliliter of a 0.25% trypsin-containing EDTA solution was added thereto, and incubated within an incubator with 5% CO 2 at 37° C. for 2 min. Then, the cells were confirmed to be peeled off.
- Ten milliliters of DMEM (containing 10% FBS) was added thereto and the cells were well-suspended.
- the dMEFs for one culture dish were seeded onto new five 10 cm culture dishes and further cultured. After 5 to 6 days of culturing, the dMEFs were confirmed to be grown confluent and washed with 6 mL of PBS. One milliliter of a 0.25% trypsin/EDTA solution was added thereto, and incubated within an incubator with 5% CO 2 at 37° C. for 2 min. Then, the cells were confirmed to be peeled off. Six milliliters of DMEM (containing 10% FBS) was added thereto and the cells were well-suspended. The resultant suspension liquid was transferred into a 50 mL tube and centrifuged at 1.4 krpm at room temperature for 4 min.
- a pMX-GFP vector is a vector in which a gene coding for a full-length GFP protein is inserted into a multi-cloning site of a pMX vector and a pMXpuro vector (obtained from The Institute of Medical Science, The University of Tokyo). Note that, the sequence of the gene coding for a full-length GFP protein is deposited in NCBI under Accession number L29345.
- a pMX-GLIS1 vector is a vector in which a gene coding for a full-length GLIS1 protein is inserted into a multi-cloning site of a pMX vector (available from Addgene). Note that, the sequence of the gene coding for a full-length GLIS1 protein is deposited in NCBI under Accession number NM_147221.
- a pMX-Neurogenin3 vector is a vector in which a gene coding for a full-length Neurogenin3 protein is inserted into a multi-cloning site of a pMX vector (obtained from The Institute of Medical Science, The University of Tokyo). Note that, the sequence of the gene coding for a full-length Neurogenin3 protein is deposited in NCBI under Accession number NM_009719.
- a pMX-Pdx1 vector is a vector in which a gene coding for a full-length Pdx1 protein is inserted into a multi-cloning site of a pMX vector (obtained from The Institute of Medical Science, The University of Tokyo). Note that, the sequence of the gene coding for a full-length Pdx1 protein is deposited in NCBI under Accession number NM_008814.
- the Plat-E cells were seeded in a 6-well plate (available from TPP, 92406), which had been coated (for 1 hour at 37° C. and 5% CO 2 ) with Poly-L-Lysine (available from Sigma, P8920) diluted 10 fold with PBS, at 8 ⁇ 10 5 cells per well, and cultured overnight.
- plasmid/OPTI-MEM solution 10 ⁇ L of LIPOFECTAMINE (registered trademark) 2000 (LP2000) (available from Life Technologies Corporation, 11668500) was placed into another 1.5 mL tube containing 250 ⁇ L of OPTI-MEM, mixed together, and left to stand at room temperature for 5 min (hereinafter may be referred to as “LP2000/OPTI-MEM solution”).
- Plasmid/OPTI-MEM solution and the LP2000/OPTI-MEM solution were well-mixed together and left to stand at room temperature for 20 min (hereinafter may be referred to as “plasmid/LP2000/OPTI-MEM mixed solution”).
- the plasmid/LP2000/OPTI-MEM mixed solution in which liposome-DNA complexes had been formed was added to one well in the 6-well plate, in which the Plat-E cells seeded the previous day had been cultured, to thereby transfect the cells. After mixing, the cells were cultured within an incubator with 5% CO 2 at 37° C. overnight. Twenty-four hours after, the medium was replaced, 1.5 mL of fresh DMEM (containing 10% FBS) was added thereto, and further cultured for 24 hours.
- the culture supernatant containing viral particles was collected in a 2.5 mL syringe (available from Terumo Corporation, SS-02SZ) and filtered through a 0.45 filter (available from Whatman, PURADISC FP30 (CA-S 0.45 ⁇ m), 10462100) to thereby remove the Plat-E cells.
- the culture supernatant containing viral particles were transferred into a 2.0 mL tube.
- a pMX-GLIS1 vector-derived viral solution a pMX-Neurogenin3 vector-derived viral solution, a pMX-Pdx1 vector-derived viral solution, and a pMX-GFP vector-derived viral solution were obtained.
- the dMEFs were infected with the retrovirus to thereby introduce the gene(s).
- the infection was performed in the following manner.
- the dMEFs were seeded in a 12-well plate at 1 ⁇ 10 5 cells per well.
- an 8 mg/mL polybrene solution (available from Sigma, 107689) was added to the culture supernatant containing viral particles, which was collected as described under the section entitled “Production of retrovirus”, at a final concentration of 8 ⁇ g/mL.
- the culture supernatant of the dMEFs was removed through aspiration, and then the below-described viral solutions were added to a 12-well plate (200 ⁇ L per well). Amounts of the viral solutions were adjusted so as to be uniform for each well with a DMEM (containing 10% FBS) solution containing 8 ⁇ g/mL polybrene.
- the resultant solutions were incubated within an incubator with 5% CO 2 at 37° C. During the incubation, the media were changed every 2 or 3 days.
- G1 solution (1) pMX-GLIS1 vector-derived viral solution (hereinafter may be referred to as “G1 solution”); (2) pMX-Neurogenin3 vector-derived viral solution (hereinafter may be referred to as “N solution”); (3) pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “P solution”); (4) pMX-Neurogenin3 vector-derived viral solution and pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “NP solution”); (5) pMX-GLIS1 vector-derived viral solution and pMX-Neurogenin3 vector-derived viral solution (hereinafter may be referred to as “G1N solution”); (6) pMX-GLIS1 vector-derived viral solution and pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “G1P solution”); (7) pMX-GLIS1 vector-derived viral solution, p
- DsRed2-positive insulin producing cells were observed and photographed by a fluorescence microscope (CARL ZEISS AXIOVERT 200M) unit.
- HOECHST 33342 (available from Life Technologies Corporation, H1399) was added to wells of a cell culture multi-well plate at a final concentration of 0.1 ⁇ g/mL and incubated within an incubator with 5% CO 2 at 37° C. for 30 min or longer. Then, images were taken in 100 fields of view for each well using a high-end cell imaging apparatus (available from Thermo Fisher Scientific Inc., ARRAYSCAN XTI) with a 10 ⁇ objective lens. The number of the DsRed2-positive insulin producing cells relative to the number of total cells was determined in the 100 fields of view.
- FIG. 1 The results of the statistical analysis performed 12 days after the introduction are presented in FIG. 1 .
- the horizontal axis represents the viral solution used, that is, the results of the G1 solution, the N solution, the P solution, the NP solution, the G1N solution, and the G1NP solution were presented from left to right.
- the vertical axis represents the number of the DsRed2-positive insulin producing cells per well.
- pancreatic endocrine cells were capable of being produced from somatic cells in large quantities by introducing (I) the GLIS1 gene and the Ngn3 gene or (II) the GLIS1 gene, the Ngn3 gene, and the Pdx1 gene into the somatic cells.
- pancreatic endocrine cells were capable of being efficiently produced from somatic cells by introducing (I) the GLIS1 gene and the Ngn3 gene or (II) the GLIS1 gene, the Ngn3 gene, and the Pdx1 gene into the somatic cells.
- Test Example 2 Production of Pancreatic Endocrine Cells from Mouse Fibroblasts-2
- the dMEFs were prepared in the same manner as in the Test Example 1.
- the pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector-derived viral solution were produced in the same manner as in the Test Example 1.
- the genes were introduced into the dMEFs by infecting the cells with the retrovirus using the G1NP solution as the viral solution in the same manner as in the Test Example 1.
- the cells were fixed with a 4% paraformaldehyde solution at room temperature for 10 min, dipped into a 0.2% Triton-X/PBS solution at room temperature for 10 min, and then treated with 4-fold diluted BLOCKING ONE solution (available from NACALAI TESQUE, INC.) at room temperature for 1 hour.
- a primary antibody reaction was performed with an anti-insulin antibody (400-fold diluted, guinea pig, available from DAKO), an anti-glucagon antibody (400-fold diluted, rabbit, available from DAKO), an anti-somatostatin antibody (500-fold diluted, rabbit, available from DAKO), or an anti-Pdx1 antibody (1,000-fold diluted, rabbit, obtained from Vanderbilt University, USA) at 4° C. overnight.
- an anti-insulin antibody 400-fold diluted, guinea pig, available from DAKO
- an anti-glucagon antibody 400-fold diluted, rabbit, available from DAKO
- an anti-somatostatin antibody 500-fold diluted, rabbit, available from DAKO
- an anti-Pdx1 antibody 1,000-fold diluted, rabbit, obtained from Vanderbilt University, USA
- the resultant reaction products were washed with PBS 3 times at room temperature for 5 min, and subjected to a secondary antibody reaction with AlexaFluor-Cy3-labeled anti-rabbit antibody (400-fold diluted, available from Invitrogen) or AlexaFluor-488-labeled anti-guinea pig antibody (400-fold diluted, available from Invitrogen) at room temperature for 1 hour.
- the resultant reaction products were washed with PBS 3 times for 5 min, and then observed and photographed by an inverted fluorescence microscope (CARL ZEISS AXIOVERT 200M).
- FIGS. 2A to 2C The results of the immunostaining analysis are presented in FIGS. 2A to 2C .
- FIG. 2A illustrates the expression analysis results of insulin and somatostatin
- FIG. 2B illustrates the expression analysis results of insulin and glucagon
- FIG. 2C illustrates the expression analysis results of insulin and Pdx1.
- the arrows with a solid line represent cells in which insulin expression is confirmed.
- the arrows with a dashed line represent cells in which somatostatin expression in FIG. 2A , glucagon expression in FIG. 2B , or Pdx1 expression in FIG. 2C is confirmed.
- Human HEK293 (cell line established by transforming human embryonic renal cells with Adenovirus E1 gene)
- the pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-GFP vector-derived viral solution were produced for mouse cells in the same manner as in the Test Example 1.
- the pMX-GFP vector was prepared in the same manner as in the Test Example 1.
- the pMX-GLIS1 vector was prepared in the same manner as in the Test Example 1, except that the sequence of the gene coding for a full-length GLIS1 protein deposited under Accession number NM_147221 was changed to the sequence of the gene coding for a full-length GLIS1 protein deposited under Accession number NM_147193.
- the pMX-Neurogenin3 vector was prepared in the same manner as in the Test Example 1, except that the sequence of the gene coding for a full-length Neurogenin3 protein deposited under Accession number NM_009719 was changed to the sequence of the gene coding for a full-length Neurogenin3 protein deposited under Accession number NM_020999.
- the pMX-Pdx1 vector was prepared in the same manner as in the Test Example 1, except that the sequence of the gene coding for a full-length Pdx1 protein deposited under Accession number NM_008814 was changed to the sequence of the gene coding for a full-length Pdx1 protein deposited under Accession number NM_000209.
- the Plat-GP cells were seeded in a 6-well plate (available from TPP, 92406), which had been coated (for 1 hour at 37° C. and 5% CO 2 ) with Poly-L-Lysine (available from Sigma, P8920) diluted 10 fold with PBS, at 8 ⁇ 10 5 cells per well, and cultured overnight.
- plasmid/OPTI-MEM solution 4 ⁇ g of the plasmid DNA (2 ⁇ g of the pMX vector and 2 ⁇ g of the VSVG vector) was placed into a 1.5 mL tube containing 250 ⁇ L of OPTI-MEM (registered trademark) (available from Life Technologies Corporation, 11058021), mixed by tapping, and left to stand at room temperature for 5 min (hereinafter may be referred to as “plasmid/OPTI-MEM solution”).
- OPTI-MEM registered trademark
- LIPOFECTAMINE (registered trademark) 2000 (LP2000) (available from Life Technologies Corporation, 11668500) was placed into another 1.5 mL tube containing 250 ⁇ L of OPTI-MEM, mixed together, and left to stand at room temperature for 5 min (hereinafter may be referred to as “LP2000/OPTI-MEM solution”).
- LP2000/OPTI-MEM solution The plasmid/OPTI-MEM solution and the LP2000/OPTI-MEM solution were well-mixed together and left to stand at room temperature for 20 min (hereinafter may be referred to as “plasmid/LP2000/OPTI-MEM mixed solution”).
- the plasmid/LP2000/OPTI-MEM mixed solution in which liposome-DNA complexes had been formed was added to one well in the 6-well plate, in which the Plat-GP cells seeded the previous day had been cultured, to thereby transfect the cells. After mixing, the cells were cultured within an incubator with 5% CO 2 at 37° C. overnight. Twenty-four hours after, the medium was replaced, 1.5 mL of fresh DMEM (containing 10% FBS) was added thereto, and further cultured for 24 hours.
- the culture supernatant containing viral particles was collected in a 2.5 mL syringe (available from Terumo Corporation, SS-02SZ) and filtered through a 0.45 filter (available from Whatman, PURADISC FP30 (CA-S 0.45 ⁇ m), 10462100) to thereby remove the Plat-GP cells.
- the culture supernatant containing viral particles were transferred into a 2.0 mL tube.
- a pMX-GLIS1 vector-derived viral solution a pMX-Neurogenin3 vector-derived viral solution, a pMX-Pdx1 vector-derived viral solution, and a pMX-GFP vector-derived viral solution for human cells were obtained.
- the genes were introduced into the cells by infecting the cells with the retrovirus using the GIN solution or the GFP CTL solution as the viral solution in the same manner as in the Test Example 1.
- a quantitative PCR analysis was performed as described below using the cells 20 days after the introduction to thereby determine a relative expression level of an insulin gene relative to a GAPDH gene.
- the cells were suspended in a cell lysis solution, and subjected to RNA preparation and cDNA synthesis using SuperPrepTM Cell Lysis & RT Kit for qPCR (available from TOYOBO CO., LTD., #SCQ-101) or SV 96 Total RNA Isolation System (available from Promega, #Z3505), ReverTraAce qPCR RT Master Mix with gDNA Remover (available from TOYOBO CO., LTD., #FSQ-301) and then the quantitative PCR analysis using GeneAce SYBR qPCR Mix ⁇ (available from NIPPON GENE CO., LTD.).
- SuperPrepTM Cell Lysis & RT Kit for qPCR available from TOYOBO CO., LTD., #SCQ-101
- SV 96 Total RNA Isolation System available from Promega, #Z3505
- ReverTraAce qPCR RT Master Mix with gDNA Remover available from TOYOBO CO., LTD., #FSQ-301
- FIGS. 3A to 3G The results of the quantitative PCR analysis are presented in FIGS. 3A to 3G .
- FIG. 3A illustrates the results of the dMEFs
- FIG. 3B illustrates the results of the human iPS (253G13-6)-derived fibroblasts
- FIG. 3C illustrates the results of the human HepG2
- FIG. 3D illustrates the results of the mouse NIH-3T3
- FIG. 3E illustrates the results of the human embryonic fibroblasts
- FIG. 3F illustrates the results of the human neonatal fibroblasts
- FIG. 3G illustrates the results of the human HEK293.
- FIGS. 3A illustrates the results of the dMEFs
- FIG. 3B illustrates the results of the human iPS (253G13-6)-derived fibroblasts
- FIG. 3C illustrates the results of the human HepG2
- FIG. 3D illustrates the results of the mouse NIH-3T3
- CTL represents the results in the case of using the GFP CTL solution
- GIN represents the results in the case of using the GIN solution.
- the vertical axis represents the relative expression level of an insulin gene relative to a GAPDH gene.
- pancreatic endocrine cells were capable of being produced from various somatic cells by the method of the present invention.
- the dMEFs were prepared in the same manner as in the Test Example 1.
- the pMX-GFP vector was prepared in the same manner as in the Test Example 1.
- the pMX-GLIS1 vector was prepared in the same manner as in the Test Example 1.
- a pMX-GLIS3 vector is a vector in which a gene coding for a full-length GLIS3 protein is inserted into a multi-cloning site of a pMX vector (obtained from The Institute of Medical Science, The University of Tokyo). Note that, the sequence of the gene coding for a full-length GLIS3 protein is deposited in NCBI under Accession number NM_175459.
- the pMX-Neurogenin3 vector was prepared in the same manner as in the Test Example 1.
- the pMX-Pdx1 vector was prepared in the same manner as in the Test Example 1.
- a culture supernatant containing viral particles was prepared in the same manner as in the Test Example 1, except that the plasmid DNA was used. The resultant culture supernatant was used as a viral solution.
- the genes were introduced into the dMEFs by infecting the cells with the retrovirus in the same manner as in the Test Example 1, except that the following viral solutions were used.
- G1 solution pMX-GFP vector-derived viral solution
- G3 solution pMX-GLIS1 vector-derived viral solution
- G1N solution pMX-GLIS1 vector-derived viral solution and pMX-Neurogenin3 vector-derived viral solution
- G1N solution pMX-GLIS3 vector-derived viral solution and pMX-Neurogenin3 vector-derived viral solution
- G1N solution pMX-GLIS3 vector-derived viral solution and pMX-Neurogenin3 vector-derived viral solution
- G3N solution pMX-GLIS3 vector-derived viral solution and pMX-Neurogenin3 vector-derived viral solution
- G3N solution pMX-GLIS1 vector-derived viral solution, pMX-Neurogenin3 vector-derived viral solution, and pMX-Pdx1 vector-derived viral solution
- the number of the DsRed2-positive insulin producing cells was counted in the same manner as in the Test Example 1, except that cells 11 days after the introduction were used.
- FIGS. 4A to 4D The counting results of the DsRed2-positive insulin producing cells are presented in FIGS. 4A to 4D .
- FIG. 4A illustrates the results in the case of using the GFP CTL solution (GFP), the G1 solution (G1), and the G1N solution (G1N) as the viral solution from left to right
- FIG. 4B illustrates the results in the case of using the GFP CTL solution (GFP), the G1 solution (G1), and the G1NP solution (G1NP) as the viral solution from left to right
- FIG. 4C illustrates the results in the case of using the GFP CTL solution (GFP), the G3 solution (G3), and the G3N solution (G3N) as the viral solution from left to right; and
- 4D illustrates the results in the case of using the GFP CTL solution (GFP), the G3 solution (G3), and the G3NP solution (G3NP) as the viral solution from left to right.
- GFP GFP CTL solution
- G3 G3 solution
- G3NP G3NP solution
- the vertical axis represents the number of the DsRed2-positive insulin producing cells per well.
- pancreatic endocrine cells were capable of being produced by introducing along with Neurogenin3, or Neurogenin3 and Pdx1 into somatic cells. Therefore, it was suggested that pancreatic endocrine cells were capable of being produced by introducing the GLIS family along with Neurogenin3, or Neurogenin3 and Pdx1 into somatic cells. It was also demonstrated from comparison of GLIS1 with the GLIS3 that the GLIS1 yields about 10 times higher transdifferentiation efficiency than the GLIS3.
- the pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector- and pMX-GFP vector-derived viral solution were produced in the same manner as Production of retrovirus for human cells in the Test Example 3.
- the genes were introduced into the cells by infecting the cells with the retrovirus using the GFP CTL solution, the G1N solution, or the G1NP solution as the viral solution in the same manner as in the Test Example 1.
- the quantitative PCR analysis was performed in the same manner as in the Test Example 3 to thereby determine a relative expression level of an insulin gene relative to a GAPDH gene.
- FIGS. 5A and 5B The results of the quantitative PCR analysis are presented in FIGS. 5A and 5B .
- FIG. 5A illustrates the results of the human T98G glioblastoma; and
- FIG. 5B illustrates the results of the human mesenchymal stem cells.
- CTL represents the results in the case of using the GFP CTL solution
- MN represents the results in the case of using the G1N solution
- G1NP represents the results in the case of using the G1NP solution.
- the vertical axis represents the relative expression level of an insulin gene relative to a GAPDH gene.
- pancreatic endocrine cells were capable of being produced from various somatic cells by the method of the present invention.
- the dMEFs were prepared in the same manner as in the Test Example 1.
- the pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector-derived viral solution were produced in the same manner as in the Test Example 1.
- the genes were introduced into the dMEFs by infecting the cells with the retrovirus using the G1NP solution as the viral solution in the same manner as in the Test Example 1.
- FIGS. 6A and 6B The results of the microscopic observation are presented in FIGS. 6A and 6B .
- FIG. 6A illustrates a pancreatic islet isolated from a mouse pancreas and
- FIG. 6B illustrates the result in the case of using the G1NP solution as the viral solution.
- pancreatic endocrine cell masses obtained by the method of the present invention resemble pancreatic islets isolated from mouse pancreas.
- the dMEFs were prepared in the same manner as in the Test Example 1.
- the pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector-derived viral solution were produced in the same manner as in the Test Example 1.
- the genes were introduced into the dMEFs by infecting the cells with the retrovirus using the G1NP solution as the viral solution in the same manner as in the Test Example 1.
- pancreatic islet-like masses were cultured in a 2.8 mM glucose-containing Ringer's solution for 3 hours. Then, the medium was replaced and the masses were cultured for another 1 hour, of which culture supernatants were used as a reference (hereinafter may be referred to as “reference culture supernatant”).
- pancreatic islet-like masses were cultured in a 16.8 mM glucose-containing Ringer's solution for 1 hour.
- a culture supernatants thereof were transferred into a 1.5 mL tube (hereinafter may be referred to as “high-glucose culture supernatant”).
- pancreatic islet-like masses were cultured for 1 hour.
- a culture supernatants thereof were transferred into a 1.5 mL tube (hereinafter may be referred to as “low-glucose culture supernatant”).
- FIG. 7 illustrates the results for each of two wells.
- (1) illustrates the result of the reference culture supernatant
- (2) illustrates the result of the high-glucose culture supernatant
- (3) illustrates the result of the low-glucose culture supernatant.
- pancreatic endocrine cells obtained by the method of the present invention were confirmed to have functions required for pancreatic endocrine cells.
- NHDF Human neonatal fibroblasts
- the pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector-derived viral solution were produced in the same manner as in Production of retrovirus for human cells in the Test Example 3.
- the genes were introduced into the human neonatal fibroblasts (NHDF) by infecting the cells with the retrovirus in the same manner as in the Test Example 1, except that the G1NP solution was used as the viral solution and a 24-well plate was used.
- pancreatic islet-like mass was picked up by a pipette and transferred into a 24-well plate (low attachment plate (EZ-BINDSHUT II, available from IWAKI)). Then, a glucose-responsive insulin secretion test was performed in the following manner.
- pancreatic islet-like mass was cultured in a 2.8 mM glucose-containing Ringer's solution for 3 hours. Then, the medium was replaced and the mass was cultured for another 1 hour, of which culture supernatant was used as a reference (hereinafter may be referred to as “reference culture supernatant”).
- pancreatic islet-like mass was cultured in a 25.0 mM glucose-containing Ringer's solution for 1 hour.
- a culture supernatant thereof was transferred into a 1.5 mL tube (hereinafter may be referred to as “high-glucose culture supernatant”).
- pancreatic endocrine cells had functions required for pancreatic endocrine cells.
- the dMEFs were prepared in the same manner as in the Test Example 1.
- a pCI-GFP vector is a vector in which a gene coding for a full-length GFP protein is inserted into a multi-cloning site of a pCI vector (available from Promega) which is an episomal vector. Note that, the sequence of the gene coding for a full-length GFP protein is deposited in NCBI under Accession number L29345.
- a pCI-GLIS1 vector is a vector in which a gene coding for a full-length GLIS1 protein is inserted into a multi-cloning site of a pCI vector (available from Promega) which is an episomal vector. Note that, the sequence of the gene coding for a full-length GLIS1 protein is deposited in NCBI under Accession number NM_147221.
- a pCI-Neurogenin3 vector is a vector in which a gene coding for a full-length Neurogenin3 protein is inserted into a multi-cloning site of a pCI vector (available from Promega) which is an episomal vector. Note that, the sequence of the gene coding for a full-length Neurogenin3 protein is deposited in NCBI under Accession number NM_009719.
- a pCI-Pdx1 vector is a vector in which a gene coding for a full-length Pdx1 protein is inserted into a multi-cloning site of a pCI vector (available from Promega) which is an episomal vector. Note that, the sequence of the gene coding for a full-length Pdx1 protein is deposited in NCBI under Accession number NM_008814.
- the following vectors were introduced into the dMEFs by electroporation using NEON (registered trademark) transfection system (available from Life Technologies Corporation). After the vectors were introduced, the cells were incubated within an incubator with 5% CO 2 at 37° C. During the incubation, the media (DMEM (containing 10% FBS)) were changed every 2 or 3 days.
- NEON registered trademark
- DMEM containing 10% FBS
- GFP pCI-GFP vector
- G1N pCI-GLIS1 vector and pCI-Neurogenin3 vector
- G1NP pCI-GLIS1 vector, pCI-Neurogenin3 vector, and pCI-Pdx1 vector
- the number of the DsRed2-positive insulin producing cells was counted in the same manner as in the Test Example 1, except that cells 8 days after the introduction were used.
- the counting results of the DsRed2-positive insulin producing cells are presented in FIG. 9 .
- the horizontal axis represents the episomal vectors introduced, that is, the results of the GFP, the G1N, and the G1NP were presented from left to right.
- the vertical axis represents the number of DsRed2-positive insulin producing cells per well.
- pancreatic endocrine cells were capable of being produced from somatic cells by introducing (I) the GLIS1 gene and the Ngn3 gene or (II) the GLIS1 gene, the Ngn3 gene, and the Pdx1 gene.
- a method for producing pancreatic endocrine cells including introducing a gene or one or more gene products thereof into somatic cells according to the present invention is simple, is easily reproduced, and has a remarkably shortened production time compared to previous methods in which pancreatic endocrine cells are produced using ES cells or iPS cells under a culturing environment properly adjusted by, for example, adding a development inhibitor to a medium. According to the method of the present invention, the pancreatic endocrine cells are capable of efficiently produced.
- the method of the present invention is also advantageous in that the pancreatic endocrine cells are capable of being produced without undergoing the iPS cell stage that have a risk of forming tumors.
- the method for producing pancreatic endocrine cells according to the present invention is suitably available for, for example, producing pancreatic endocrine cells to be used in regenerative therapies for diabetes.
- a method for producing pancreatic endocrine cells including introducing one or more genes of a GLIS family or one or more gene products thereof and a Neurogenin3 gene or one or more gene products thereof into somatic cells.
- the introducing includes further introducing a Pdx1 gene or one or more gene products thereof into the somatic cells.
- the one or more genes of the GLIS family or one or more gene products thereof are a GLIS1 gene or one or more gene products thereof.
- ⁇ 4> The method for producing pancreatic endocrine cells according to any one of ⁇ 1> to ⁇ 3>, wherein the somatic cells are fibroblasts or mesenchymal stem cells.
- ⁇ 5> The method for producing pancreatic endocrine cells according to any one of ⁇ 1> to ⁇ 4>, wherein the pancreatic endocrine cells are f3 cells.
- ⁇ 6> Pancreatic endocrine cells produced by the method for producing pancreatic endocrine cells according to any one of ⁇ 1> to ⁇ 5>.
- ⁇ 7> The pancreatic endocrine cells according to ⁇ 6>, wherein the pancreatic endocrine cells include ⁇ cells.
- a transdifferentiation agent including:
- transdifferentiation agent is configured to transdifferentiate somatic cells into pancreatic endocrine cells.
- the transdifferentiation agent according to ⁇ 8> further including a Pdx1 gene or one or more gene products thereof.
- the transdifferentiation agent according to ⁇ 8> or ⁇ 9> wherein the one or more genes of the GLIS family or one or more gene products thereof is a GLIS1 gene or one or more gene products thereof.
- the somatic cells are fibroblasts or mesenchymal stem cells.
- the pancreatic endocrine cells are ⁇ cells.
Abstract
-
- introducing one or more genes of a GLIS family or one or more gene products thereof and a Neurogenin3 gene or one or more gene products thereof into somatic cells.
Description
- The present invention relates to a method for producing pancreatic endocrine cells from somatic cells, pancreatic endocrine cells produced by the method, and a transdifferentiation agent that transdifferentiates somatic cells to pancreatic endocrine cells.
- Pancreatic endocrine cells have been expected to be used as, for example, a material for regenerative therapies for diabetes or a material used for screening of diabetes drugs. In terms of the regenerative therapies, for example, it has been expected that β cells, which are one of the pancreatic endocrine cells and produce insulin, are administered to type I diabetes patients who are insulin-deficient.
- Therefore, keen demand has arisen for developing a method for preparing pancreatic endocrine cells in vitro in large quantities.
- There has been proposed a method for producing β cells using embryonic stem cells (hereinafter may be referred to as “ES cells”) or induced pluripotent stem cells (hereinafter may be referred to as “iPS cells”). However, the method has the following problems. Firstly, the method is complicated because culturing environments are needed to be properly adjusted by, for example, adding various inhibitors involved in development or differentiation to a cell culture medium. Secondly, the method may be unreproducible. Thirdly, the method is problematic in terms of efficiency because other cells than the β cells are also produced. Finally, the method takes at least 21 days to 30 days to produce the β cells, that is, the β cells are not capable of being produced in a short period of time.
- Therefore, at present, keen demand has arisen for promptly providing a method for producing pancreatic endocrine cells, the method being simple, easily reproduced, excellent in production efficiency, and capable of producing the pancreatic endocrine cells in a short period of time.
- Note that, GLIS1 (GMS family zinc finger 1), which is a member of the GUS family, has been known to improve an establishment improving efficiency of iPS cells (see, e.g., PTL 1). GLIS3 (GLIS family zinc finger 3) has been known to be capable of being used for inducing differentiation of human pluripotent or multipotent cells into functional pancreatic β cells that produce insulin (see, e.g., PTL 2). Ngn3 (Neurogenin3) has been known to be transiently expressed in pancreatic endocrine cells during pancreas development.
- However, it has not been that the GUS family or the Ngn3 transform somatic cells into pancreatic endocrine cells directly without undergoing the stem cell stage.
- The present invention aims to solve the above existing problems and achieve the following object. That is, the present invention has an object to provide a method for producing pancreatic endocrine cells, the method being simple, easily reproduced, excellent in production efficiency, and capable of producing the pancreatic endocrine cells in a short period of time; pancreatic endocrine cells produced by the method; and a transdifferentiation agent that transdifferentiates somatic cells to pancreatic endocrine cells.
- Means for solving the above problems are as follows.
- <1> A method for producing pancreatic endocrine cells, the method including
- introducing one or more genes of a GUS family or one or more gene products thereof and a Neurogenin3 gene or one or more gene products thereof into somatic cells.
- <2> Pancreatic endocrine cells produced by the method for producing pancreatic endocrine cells according to <1>.
<3> A transdifferentiation agent including: - one or more genes of a GUS family or one or more gene products thereof, and
- a Neurogenin3 gene or one or more gene products thereof,
- wherein the transdifferentiation agent is configured to transdifferentiate somatic cells into pancreatic endocrine cells.
- According to the present invention, it is possible to solve the above existing problems and achieve the above object. That is, the present invention can provide a method for producing pancreatic endocrine cells, the method being simple, easily reproduced, excellent in production efficiency, and capable of producing the pancreatic endocrine cells in a short period of time; pancreatic endocrine cells produced by the method; and a transdifferentiation agent that transdifferentiates somatic cells to pancreatic endocrine cells.
-
FIG. 1 is a graph illustrating the measurement results of the number of DsRed2-positive insulin producing cells 12 days after viral infection in Test Examples 1. -
FIG. 2A is an image illustrating the analysis results of expression of insulin and somatostatin in Test Examples 2. -
FIG. 2B is an image illustrating the analysis results of expression of insulin and glucagon in Test Examples 2. -
FIG. 2C is an image illustrating the analysis results of expression of insulin and Pdx1 in Test Examples 2. -
FIG. 3A is a graph illustrating the results in dMEFs in Test Example 3. -
FIG. 3B is a graph illustrating the results in human iPS (253G13-6)-derived fibroblasts in Test Example 3. -
FIG. 3C is a graph illustrating the results in human HepG2 in Test Example 3. -
FIG. 3D is a graph illustrating the results in mouse NIH-3T3 in Test Example 3. -
FIG. 3E is a graph illustrating the results in human embryonic fibroblasts in Test Example 3. -
FIG. 3F is a graph illustrating the results in human neonatal fibroblasts in Test Example 3. -
FIG. 3G is a graph illustrating the results in human HEK293 in Test Example 3. -
FIG. 4A is a graph-1 illustrating the results in Test Example 4. -
FIG. 4B is a graph-2 illustrating the results in Test Example 4. -
FIG. 4C is a graph-3 illustrating the results in Test Example 4. -
FIG. 4D is a graph-4 illustrating the results in Test Example 4. -
FIG. 5A is a graph illustrating the results in human T98G glioblastoma in Test Example 5. -
FIG. 5B is a graph illustrating the results in human mesenchymal stem cells in Test Example 5. -
FIG. 6A is an image illustrating a pancreatic islet isolated from a mouse pancreas in Test Example 6. -
FIG. 6B is an image illustrating dMEFs infected with a retrovirus using a G1NP solution as a viral solution in Test Example 6. -
FIG. 7 is a graph illustrating the results of a glucose-responsive insulin secretion test in Test Example 7. -
FIG. 8 is a graph illustrating the results of a glucose-responsive insulin secretion test in Test Example 8. -
FIG. 9 is a graph illustrating the results in Test Example 9. - Pancreatic endocrine cells of the present invention are capable of being produced by a method for producing pancreatic endocrine cells of the present invention.
- The pancreatic endocrine cells of the present invention will now be described in conjunction with the method for producing pancreatic endocrine cells of the present invention.
- The method for producing pancreatic endocrine cells of the present invention includes at least an introduction step; and, if necessary, further includes other steps.
- The introduction step is a step of introducing one or more genes of a GLIS family or one or more gene products thereof and an Ngn3 gene or one or more gene products thereof into somatic cells.
- The gene products refer to mRNAs transcribed from genes or proteins translated from the mRNAs.
- The genes or one or more gene products thereof to be introduced into the somatic cells in the introduction step include at least one or more genes of a GLIS family or one or more gene products thereof and an Ngn3 gene or one or more gene products thereof; and, if necessary, further include other genes or one or more gene products thereof.
- A source of the one or more genes of the GLIS family is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include human and mouse.
- The one or more genes of the GLIS family are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include GLIS1, GLIS2, and GLIS3. These may be used alone or in combination. Among the one or more genes of the GLIS family, GLIS1 and GLIS3 are preferable, and GLIS1 is more preferable from the viewpoint of excellent production efficiency of the pancreatic endocrine cells.
- Sequence information of the one or more genes of the GLIS family is available from known databases. For example, the sequence information is available from NCBI under Accession numbers of NM_147193 (human GLIS1), NM_147221 (mouse GLIS1), NM_032575 (human GLIS2), NM_031184 (mouse GLIS2), NM_152629 (human GLIS3), NM_175459, and NM_172636 (mouse GLIS3).
- A source of the Ngn3 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include human and mouse.
- Sequence information of the Ngn3 gene is available from known databases. For example, the sequence information is available from NCBI under Accession numbers of NM_009719 (mouse) and NM_020999 (human).
- The other genes or one or more gene products thereof are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they do not impair effects of the present invention. A Pdx1 gene or one or more gene products thereof are preferable.
- A source of the Pdx1 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include human and mouse.
- Sequence information of the Pdx1 gene is available from known databases. For example, the sequence information is available from NCBI under Accession numbers of NM_000209 (human) and NM_008814 (mouse).
- Each of sequences of the one or more genes of the GUS family or one or more gene products thereof, the Ngn3 gene or one or more gene products thereof, and the other genes or one or more gene products thereof may consist of a region to be translated into a protein in the sequence of each of the genes, or may include other regions than the region to be translated into a protein. Each of the genes or one or more gene products thereof may have a mutation.
- Examples of the mutation include mutations that do not change an amino acid sequence of a protein from each of the genes and mutations in which one or several (2 to 5) amino acids are deleted, substituted, inserted, or added in an amino acid sequence of a protein from each of the genes.
- In the case where each of the genes or one or more gene products thereof has a mutation, a sequence homology to each of corresponding wild-type genes or one or more gene products thereof is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more in a base sequence of the region to be translated into a protein.
- The genes or one or more gene products thereof to be introduced into the somatic cells in the introduction step are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they include at least the one or more genes of the GUIS family or one or more gene products thereof and the Ngn3 gene or one or more gene products thereof. However, the genes or one or more gene products thereof preferably consist of (1) the one or more genes of the GUS family or one or more gene products thereof and the Ngn3 gene or one or more gene products thereof or (2) the one or more genes of the GUS family or one or more gene products thereof, the Ngn3 gene or one or more gene products thereof, and the Pdx1 gene or one or more gene products thereof, from the viewpoints of higher simplicity, easiness of reproduction, excellent production efficiency, and production of the pancreatic endocrine cells in a short period of time.
- —Somatic Cells—The somatic cells are not particularly limited and may be appropriately selected depending on the intended purpose. The somatic cells may be undifferentiated precursor cells or terminally differentiated mature cells.
- The somatic cells may be derived from ES cells or iPS cells.
- Specific examples of the somatic cells include adipose tissue-derived interstitial (stem) cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, fibroblasts, hepatic cells, epithelial cells, renal cells, macrophages, lymphocytes, and muscle cells. Among them, fibroblasts, mesenchymal stem cells, hepatic cells, epithelial cells, and renal cells are preferable, and fibroblasts and mesenchymal stem cells are more preferable.
- A species of an individual from which the somatic cells are harvested is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include human and mouse.
- An individual from which the somatic cells are harvested is not particularly limited and may be appropriately selected depending on the intended purpose. In the case where the resultant pancreatic endocrine cells are used for regenerative therapies, the individual is preferably the individual oneself or other individuals having the same or substantially the same MHC type as that of the individual, in terms of a rejection reaction. The phrase “substantially the same MHC type” means, as used herein, that the MHC type is compatible to the extent that, when pancreatic endocrine cells derived from the somatic cells are transplanted into an individual, transplanted cells are capable of being engrafted with the use of, for example, an immunosuppressive agent.
- A time when the somatic cells are harvested from the individual is not particularly limited and may be appropriately selected depending on the intended purpose.
- A condition under which the somatic cells are cultured is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a culturing temperature of about 37° C. and a CO2 concentration of from about 2% to about 5%.
- A medium in which the somatic cells are cultured is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include minimum essential media (hereinafter may be referred to as “MEM”), Dulbecco's modified Eagle media (hereinafter may be referred to as “DMEM”), RPMI1640 media, 199 media, and F12 media, all of which contain from 5% by mass to 20% by mass of serum.
- A method for introducing each of the genes or one or more gene products thereof into the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose. For example, vectors, synthetic mRNA (messenger RNA), or recombinant proteins may be used.
- The vector is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include viral vectors and non-viral vectors.
- Specific examples of the viral vectors include retroviral vectors and lentiviral vectors.
- Specific examples of the non-viral vectors include plasmid vectors and episomal vectors.
- A method for introducing the vector into the somatic cells is not particularly limited and may be appropriately selected from known methods in the art.
- In the case where the retroviral vectors are used, the methods described in, for example, WO 2007/69666; Cell, 126, 663-676 (2006); or Cell, 131, 861-872 (2007) may be used. In the case where the lentiviral vectors are used, the methods described in, for example, Science, 318, 1917-1920 (2007).
- In the case where the plasmid vectors are used, the methods described in, for example, Science, 322, 949-953 (2008). In the case where the episomal vectors are used, the methods described in, for example, Science, 324: 797-801 (2009) or Biochemical and Biophysical Research Communications, 426: 141-147 (2012).
- In the case where the viral vectors are used, viral particles obtained using packaging cells may be used.
- The packaging cells are cells into which viral structural protein-coding genes have been introduced. When a recombinant viral vector into which a target gene has been incorporated is introduced into the cells, recombinant viral particles into which the target gene has been incorporated are produced.
- The packaging cells are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include packaging cells based on human kidney-derived HEK293 cells or mouse fibroblast-derived NIH3T3 cells; packaging cells Platinum-E (hereinafter may be referred to as “Plat-E cells”) which are capable of producing high titer viruses for a long period of time and in which viral structural proteins gag-pol and env are expressed under the control of MoMuLV (Moloney Murine Leukemia Virus) LTR (long terminal repeats); PLAT-A cells that are designed to express Amphotropic virus-derived envelope glycoproteins; and PLAT-GP cells that are designed to express vesicular stomatitis virus-derived envelope glycoproteins.
- A method for introducing the viral vector into the packaging cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include lipofection methods, electroporation methods, and calcium phosphate methods.
- A method for infecting the somatic cells with the resultant viral particles is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include polybrene methods.
- The vector may include a marker gene for verifying whether each of the genes has been successfully introduced.
- The marker gene refers to a gene that allows for cell sorting or cell selection by introducing the marker gene into a cell. Specific examples of the marker gene include drug resistant genes, fluorescent protein genes, luminescent enzyme genes, and coloring enzyme genes. These may be used alone or in combination.
- Specific examples of the drug resistant genes include neomycin resistant genes, tetracycline resistant genes, kanamycin resistant genes, zeocin resistant genes, and hygromycin resistant genes.
- Specific examples of the fluorescent protein genes include green fluorescent protein (GFP) genes, yellow fluorescent protein (YFP) genes, and red fluorescent protein (RFP) genes.
- Specific examples of the luminescent enzyme gene include luciferase genes.
- Specific examples of the coloring enzyme genes include β galactosidase genes, β glucuronidase genes, and alkaline phosphatase genes.
- In a method for introducing each of the genes into the somatic cells using the vector, one gene may be incorporated into one vector, or two or more genes may be incorporated into one vector. By incorporating two or more genes into one vector, the two or more genes may be expressed at the same time (hereinafter may be referred to as “co-expression”).
- A method for incorporating two or more genes into one vector is not particularly limited and may be appropriately selected depending on the intended purpose. However, the two or more genes are preferably incorporated via a linkage sequence.
- The linkage sequence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include gene sequences coding for a foot and mouth disease virus (Picornaviridae Aphthovirus)-derived 2A peptide and IRESs (internal ribosome entry sites).
- A method for introducing the mRNA into the somatic cells is not particularly limited and may be appropriately selected from known methods in the art.
- A method for introducing the recombinant protein into the somatic cells is not particularly limited and may be appropriately selected from known methods in the art.
- The number of times of introduction of each of the genes or one or more gene products thereof into the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose. For example, each of the genes or one or more gene products thereof may be introduced once or two or more times.
- A time when each of the genes or one or more gene products thereof are introduced into the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose. The genes or one or more gene products thereof may be introduced at the same time or at different times.
- An amount of each of the genes or one or more gene products thereof to be introduced into the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose. The genes or one or more gene products thereof may be introduced in an equal amount or different amounts.
- The genes or one or more gene products thereof to be used may be the genes only, the gene products only, or both of the genes and the gene products.
- The genes or one or more gene products thereof may be combined with a different gene or one or more gene products thereof. The combination is not particularly limited and may be appropriately selected depending on the intended purpose. The same combination or different combinations may be used for each of the genes or one or more gene products thereof.
- In the introduction step of the genes or one or more gene products thereof, other materials than the genes or one or more gene products thereof may be introduced, so long as they do not impair effects of the present invention.
- The other steps are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they do not impair effects of the present invention. Examples thereof include a genes or genes products thereof-introduced cells culturing step in which somatic cells, into which each of the genes or one or more gene products thereof has been introduced, are cultured.
- The genes or genes products thereof-introduced cells culturing step is a step of culturing somatic cells into which each of the genes or one or more gene products thereof has been introduced.
- A condition under which the genes or genes products thereof-introduced cells are cultured is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a culturing temperature of about 37° C. and a CO2 concentration of from about 2% to about 5%.
- A medium used for culturing the genes or genes products thereof-introduced cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include MEM media, DMEM media, RPMI1640 media, 199 media, and F12 media, all of which contain from 5% by mass to 20% by mass of serum.
- A period of time of the genes or genes products thereof-introduced cells culturing step is not particularly limited and may be appropriately selected depending on the intended purpose.
- An exchange frequency of the medium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include every 2 days to 3 days.
- A method for verifying whether pancreatic endocrine cells are successfully produced by the method for producing pancreatic endocrine cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method in which expression of proteins to be expressed in the pancreatic endocrine cells is verified and a method in which expression of genes to be expressed in the pancreatic endocrine cells is verified.
- For example, whether α cells of the pancreatic endocrine cells are produced is capable of being verified by the presence or absence of glucagon expression, whether β cells of the pancreatic endocrine cells are produced is capable of being verified by the presence or absence of insulin expression, and whether δ cells of the pancreatic endocrine cells are produced is capable of being verified by the presence or absence of somatostatin expression.
- The method in which expression of proteins is verified is not particularly limited and may be appropriately selected from known methods in the art. Examples thereof include immunostaining analyses.
- The method in which expression of genes is verified is not particularly limited and may be appropriately selected from known methods in the art. Examples thereof include quantitative PCR analyses.
- According to the method for producing pancreatic endocrine cells of the present invention, the pancreatic endocrine cells are capable of being produced from somatic cells through transdifferentiation. Therefore, the method is advantageous in that the pancreatic endocrine cells are capable of being produced without undergoing the iPS cell stage that have a risk of forming tumors.
- Note that, the transdifferentiation refers to direct transformation from a cell type to another cell type without undergoing the stem cell stage.
- The method for producing pancreatic endocrine cells of the present invention is simple and easily reproduced because a gene or one or more gene products thereof only have to be introduced into somatic cells, and at the same time the pancreatic endocrine cells are capable of being produced efficiently in a short period of time. Moreover, the method for producing pancreatic endocrine cells of the present invention is also advantageous in that the pancreatic endocrine cells are capable of being produced without using a special medium of which culturing environments are properly adjusted by, for example, adding inhibitors involved in development to the medium.
- The pancreatic endocrine cells may be α cells, β cells, δ cells, or mixtures thereof. Among them, β cells are preferable in terms of regenerative therapies for diabetes patients.
- The pancreatic endocrine cells of the present invention are suitably available as pancreatic endocrine cells used for screening of diabetes drugs.
- A transdifferentiation agent of the present invention is a transdifferentiation agent for transdifferentiating somatic cells into pancreatic endocrine cells. The transdifferentiation agent includes at least one or more genes of a GUIS family or one or more gene products thereof and an Ngn3 gene or one or more gene products thereof; and, if necessary, further includes other components.
- Somatic cells to be targeted by the transdifferentiation agent and preferable aspects thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- Pancreatic endocrine cells obtained using the transdifferentiation agent and preferable aspects thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- The one or more genes of the GUS family or one or more gene products thereof and preferable aspects thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.” The one or more genes of the GUTS family or one or more gene products thereof may also have the same mutation as those described under the section entitled “Production method of pancreatic endocrine cells.”
- An aspect of the one or more genes of the GUIS family or one or more gene products thereof in the transdifferentiation agent is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the one or more genes of the GUS family or one or more gene products thereof may be incorporated into a vector, or may be a synthetic mRNA or a recombinant protein.
- The vector may be the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- The synthetic mRNA and the recombinant protein may be produced by any of known methods in the art.
- The Ngn3 gene or one or more gene products thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.” The Ngn3 gene or one or more gene products thereof may also have the same mutation as those described under the section entitled “Production method of pancreatic endocrine cells.”
- An aspect of the Ngn3 gene or one or more gene products thereof in the transdifferentiation agent is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the Ngn3 gene or one or more gene products thereof may be incorporated into a vector, or may be a synthetic mRNA or a recombinant protein.
- The vector may be the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- The synthetic mRNA and the recombinant protein may be produced by any of known methods in the art.
- The other components are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they do not impair effects of the present invention. However, the other components preferably include a Pdx1 gene or one or more gene products thereof.
- The Pdx1 gene or one or more gene products thereof are the same as those described under the section entitled “Production method of pancreatic endocrine cells.” The Pdx1 gene or one or more gene products thereof may also have the same mutation those described under the section entitled “Production method of pancreatic endocrine cells.”
- An aspect of the Pdx1 gene or one or more gene products thereof in the transdifferentiation agent is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the Pdx1 gene or one or more gene products thereof may be incorporated into a vector, or may be a synthetic mRNA or a recombinant protein.
- The vector may be the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- The synthetic mRNA and the recombinant protein may be produced by any of known methods in the art.
- The genes or one or more gene products thereof in the transdifferentiation agent may be divided into separate containers or may be placed in a single container. Alternatively, any number of the genes or one or more gene products thereof may be placed in each container.
- An amount of each of the genes or one or more gene products thereof in the transdifferentiation agent is not particularly limited. The genes or one or more gene products thereof may be included in an equal amount or different amounts.
- The transdifferentiation agent may be suitably used as a component of a kit for producing pancreatic endocrine cells.
- The kit for producing pancreatic endocrine cells includes at least the transdifferentiation agent; and, if necessary, further includes other components.
- The other components in the kit for producing pancreatic endocrine cells are not particularly limited and may be appropriately selected depending on the intended purpose, so long as they do not impair effects of the present invention. Examples thereof include packaging cells and media.
- The packaging cells and the media may be the same as those described under the section entitled “Production method of pancreatic endocrine cells.”
- The present invention will now be described with reference to Test Examples described below, but the present invention is not limited thereto in any way.
- Dual-labeled-mouse embryonic fibroblasts (hereinafter may be referred to as “dMEF”), which were one kind of somatic cells, were prepared in the following manner.
- —Production of Genetically Modified Mice in which Pancreatic Endocrine Precursor Cells are Fluorescently Labeled with GFP—
- Genetically modified mice in which pancreatic endocrine precursor cells are fluorescently labeled with GFP (mice expressing EGFP under the control of an Ngn3 gene promoter (Ngn3-eGFP)) were produced in the following manner.
- A construct, in which a fusion protein gene of GFP and a nuclear localization signal (hereinafter may be referred to as “nls”) was ligated downstream of the Ngn3 gene promoter (5 kb) isolated from a BAC clone, was microinjected into about 400 fertilized eggs to thereby produce genetically modified mice in which pancreatic endocrine precursor cells are fluorescently labeled with GFP.
- —Production of Genetically Modified Mice in which Pancreatic β Cells are Fluorescently Labeled with DsRed2—
- Genetically modified mice in which pancreatic β cells are fluorescently labeled with DsRed2 (mice expressing DsRed2 under the control of a rat insulin promoter (Ins-DsR)) were produced in the following manner.
- A construct, in which a DsRed2 gene was ligated downstream of the rat insulin promoter (800 bp), was microinjected into about 400 fertilized eggs to thereby produce genetically modified mice in which pancreatic β cells are fluorescently labeled with DsRed2.
- The genetically modified mice in which pancreatic endocrine precursor cells are fluorescently labeled with GFP were crossed with the genetically modified mice in which pancreatic β cells are fluorescently labeled with DsRed2, and then male and female offspring mice (heterozygous) were crossed with each other to generate dual-labeled genetically modified mice (Ngn3-eGFP/Ins-DsR) that were confirmed to be homozygous by genomic southern blotting. Two pairs (male and female) of the homozygous dual-labeled genetically modified mice were crossed. At embryonic day 14.5, 16 embryos were removed from the uterus, and their blood was washed off with 10 mL of phosphate-buffered saline (containing 10 mg/mL kanamycin) in a 10 cm Petri dish within a clean bench. Then, the embryos were minced with a pair of scissors in 10 mL of DMEM (available from Sigma, #D5796; containing penicillin, streptomycin, and 10% FBS) in a 10 cm cell culture dish (available from TPP, #93150). The resultant minced embryonic tissue was transferred into a 15 mL tube and centrifuged at 1.4 krpm at room temperature for 4 min. The supernatant was discarded. The remaining pellet was added with and suspended in 1 mL of a 0.25% trypsin-containing EDTA solution (available from Wako Pure Chemical Industries, Ltd., #201-16945, containing 0.25% DNase I), and then incubated in a water bath at 37° C. The water bath was stirred by hand every 10 min. The minced embryonic tissue corresponding to one animal was well-suspended in 5 mL of DMEM (containing 10% FBS) in a 15 mL tube, transferred into 5 mL of DMEM in a 10 cm cell culture dish, and then incubated within an incubator with 5% CO2 at 37° C. On the following day, the 10 mL DMEM (containing 10% FBS) was replaced with fresh medium and subsequently changed every 2 days. About 4 to about 5 days after, dMEFs in the confluent 10 cm culture dish were washed with 6 mL of phosphate-buffered saline (hereinafter may be referred to as “PBS”). One milliliter of a 0.25% trypsin-containing EDTA solution was added thereto, and incubated within an incubator with 5% CO2 at 37° C. for 2 min. Then, the cells were confirmed to be peeled off. Ten milliliters of DMEM (containing 10% FBS) was added thereto and the cells were well-suspended. The dMEFs for one culture dish were seeded onto new five 10 cm culture dishes and further cultured. After 5 to 6 days of culturing, the dMEFs were confirmed to be grown confluent and washed with 6 mL of PBS. One milliliter of a 0.25% trypsin/EDTA solution was added thereto, and incubated within an incubator with 5% CO2 at 37° C. for 2 min. Then, the cells were confirmed to be peeled off. Six milliliters of DMEM (containing 10% FBS) was added thereto and the cells were well-suspended. The resultant suspension liquid was transferred into a 50 mL tube and centrifuged at 1.4 krpm at room temperature for 4 min. Then, the supernatant was discarded. The remaining cell pellet was added with and suspended in 10 mL of CELLBANKER (available from Takara Bio Inc., #CB011). The resultant suspension liquid was dispensed into vial tubes (0.5 mL per tube) and stored in a deep freezer at −145° C.
- Plat-E cells in which viral structural proteins gag-pol and env, which were capable of producing high titer viruses for a long period of time, were expressed under the control of MoMuLV LTR and a plasmid DNA (pMX vector or pMX-puro vector) were used to produce a retrovirus in the following manner (Onishi, M., et. al., Exp. Hematol. 24, 324-329, 1996).
- [pMX-GFP Vector]
- A pMX-GFP vector is a vector in which a gene coding for a full-length GFP protein is inserted into a multi-cloning site of a pMX vector and a pMXpuro vector (obtained from The Institute of Medical Science, The University of Tokyo). Note that, the sequence of the gene coding for a full-length GFP protein is deposited in NCBI under Accession number L29345.
- [pMX-GLIS1 Vector]
- A pMX-GLIS1 vector is a vector in which a gene coding for a full-length GLIS1 protein is inserted into a multi-cloning site of a pMX vector (available from Addgene). Note that, the sequence of the gene coding for a full-length GLIS1 protein is deposited in NCBI under Accession number NM_147221.
- [pMX-Neurogenin3 Vector]
- A pMX-Neurogenin3 vector is a vector in which a gene coding for a full-length Neurogenin3 protein is inserted into a multi-cloning site of a pMX vector (obtained from The Institute of Medical Science, The University of Tokyo). Note that, the sequence of the gene coding for a full-length Neurogenin3 protein is deposited in NCBI under Accession number NM_009719.
- [pMX-Pdx1 Vector]
- A pMX-Pdx1 vector is a vector in which a gene coding for a full-length Pdx1 protein is inserted into a multi-cloning site of a pMX vector (obtained from The Institute of Medical Science, The University of Tokyo). Note that, the sequence of the gene coding for a full-length Pdx1 protein is deposited in NCBI under Accession number NM_008814.
- The Plat-E cells were seeded in a 6-well plate (available from TPP, 92406), which had been coated (for 1 hour at 37° C. and 5% CO2) with Poly-L-Lysine (available from Sigma, P8920) diluted 10 fold with PBS, at 8×105 cells per well, and cultured overnight.
- On the following day, 4 μg of the plasmid DNA was placed into a 1.5 mL tube containing 250 μL of OPTI-MEM (registered trademark) (available from Life Technologies Corporation, 11058021), mixed by tapping, and left to stand at room temperature for 5 min (hereinafter may be referred to as “plasmid/OPTI-MEM solution”). Meanwhile, 10 μL of LIPOFECTAMINE (registered trademark) 2000 (LP2000) (available from Life Technologies Corporation, 11668500) was placed into another 1.5 mL tube containing 250 μL of OPTI-MEM, mixed together, and left to stand at room temperature for 5 min (hereinafter may be referred to as “LP2000/OPTI-MEM solution”). The plasmid/OPTI-MEM solution and the LP2000/OPTI-MEM solution were well-mixed together and left to stand at room temperature for 20 min (hereinafter may be referred to as “plasmid/LP2000/OPTI-MEM mixed solution”).
- The plasmid/LP2000/OPTI-MEM mixed solution in which liposome-DNA complexes had been formed was added to one well in the 6-well plate, in which the Plat-E cells seeded the previous day had been cultured, to thereby transfect the cells. After mixing, the cells were cultured within an incubator with 5% CO2 at 37° C. overnight. Twenty-four hours after, the medium was replaced, 1.5 mL of fresh DMEM (containing 10% FBS) was added thereto, and further cultured for 24 hours.
- Forty-eight hours after the transfection, the culture supernatant containing viral particles was collected in a 2.5 mL syringe (available from Terumo Corporation, SS-02SZ) and filtered through a 0.45 filter (available from Whatman, PURADISC FP30 (CA-S 0.45 μm), 10462100) to thereby remove the Plat-E cells. The culture supernatant containing viral particles were transferred into a 2.0 mL tube.
- Thus, a pMX-GLIS1 vector-derived viral solution, a pMX-Neurogenin3 vector-derived viral solution, a pMX-Pdx1 vector-derived viral solution, and a pMX-GFP vector-derived viral solution were obtained.
- The dMEFs were infected with the retrovirus to thereby introduce the gene(s). The infection was performed in the following manner.
- The dMEFs were seeded in a 12-well plate at 1×105 cells per well.
- On the following day, an 8 mg/mL polybrene solution (available from Sigma, 107689) was added to the culture supernatant containing viral particles, which was collected as described under the section entitled “Production of retrovirus”, at a final concentration of 8 μg/mL. The culture supernatant of the dMEFs was removed through aspiration, and then the below-described viral solutions were added to a 12-well plate (200 μL per well). Amounts of the viral solutions were adjusted so as to be uniform for each well with a DMEM (containing 10% FBS) solution containing 8 μg/mL polybrene. After the addition of the viral solutions, the resultant solutions were incubated within an incubator with 5% CO2 at 37° C. During the incubation, the media were changed every 2 or 3 days.
- (1) pMX-GLIS1 vector-derived viral solution (hereinafter may be referred to as “G1 solution”);
(2) pMX-Neurogenin3 vector-derived viral solution (hereinafter may be referred to as “N solution”);
(3) pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “P solution”);
(4) pMX-Neurogenin3 vector-derived viral solution and pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “NP solution”);
(5) pMX-GLIS1 vector-derived viral solution and pMX-Neurogenin3 vector-derived viral solution (hereinafter may be referred to as “G1N solution”);
(6) pMX-GLIS1 vector-derived viral solution and pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “G1P solution”);
(7) pMX-GLIS1 vector-derived viral solution, pMX-Neurogenin3 vector-derived viral solution, and pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “G1NP solution”); and
(8) pMX-GFP vector-derived viral solution (control; hereinafter may be referred to as “GFP CTL solution”).
<Observation of dMEF-Derived Insulin Producing Cells and Counting of Number of Cells> - After the introduction and several days of culturing, DsRed2-positive insulin producing cells were observed and photographed by a fluorescence microscope (CARL ZEISS AXIOVERT 200M) unit.
- A statistical analysis was performed in the following manner. HOECHST 33342 (available from Life Technologies Corporation, H1399) was added to wells of a cell culture multi-well plate at a final concentration of 0.1 μg/mL and incubated within an incubator with 5% CO2 at 37° C. for 30 min or longer. Then, images were taken in 100 fields of view for each well using a high-end cell imaging apparatus (available from Thermo Fisher Scientific Inc., ARRAYSCAN XTI) with a 10× objective lens. The number of the DsRed2-positive insulin producing cells relative to the number of total cells was determined in the 100 fields of view.
- Two days after the introduction, observation was made by a fluorescence microscope (CARL ZEISS AXIOVERT 200M) unit. In the case where the G1N solution or the G1NP solution was used as the viral solution, fluorescence from DsRed2 was observed. Therefore, it was demonstrated that β cells, which are pancreatic endocrine cells, were capable of being produced from fibroblasts in a short period of time of 2 days when the G1N solution or the G1NP solution was used as the viral solution.
- The results of the statistical analysis performed 12 days after the introduction are presented in
FIG. 1 . InFIG. 1 , the horizontal axis represents the viral solution used, that is, the results of the G1 solution, the N solution, the P solution, the NP solution, the G1N solution, and the G1NP solution were presented from left to right. Note that, the vertical axis represents the number of the DsRed2-positive insulin producing cells per well. - It can be seen from the results of
FIG. 1 that the number of the β cells, which were pancreatic endocrine cells, was significantly increased in the case where (I) the GLIS1 gene and the Ngn3 gene or (II) the GLIS1 gene, the Ngn3 gene, and the Pdx1 gene were introduced into somatic cells. Therefore, it was demonstrated that pancreatic endocrine cells were capable of being produced from somatic cells in large quantities by introducing (I) the GLIS1 gene and the Ngn3 gene or (II) the GLIS1 gene, the Ngn3 gene, and the Pdx1 gene into the somatic cells. - The results of the statistical analysis performed 17 days after the introduction are presented in Table 1.
-
TABLE 1 Rate of number of Number of DsRed2-positive DsRed2-positive insulin producing insulin producing cells relative to cells/well number of total cells Viral G1 solution 169 0.12% solution N solution 802 1.2% P solution 89 0.09% NP solution 479 0.78% G1N solution 11809 11.8% G1P solution 78 0.07% G1NP solution-1 7055 8.5% GFP CTL solution 106 0.17% G1NP solution-2 6554 7.81% No viral infection 5 0.01% - It can be seen from the results of Table 1 that the rate of the number of the β cells, which were pancreatic endocrine cells, relative to the number of total cells was increased in the case where (I) the GLIS1 gene and the Ngn3 gene or (II) the GLIS1 gene, the Ngn3 gene, and the Pdx1 gene were introduced into somatic cells. Therefore, it was demonstrated that pancreatic endocrine cells were capable of being efficiently produced from somatic cells by introducing (I) the GLIS1 gene and the Ngn3 gene or (II) the GLIS1 gene, the Ngn3 gene, and the Pdx1 gene into the somatic cells.
- The dMEFs were prepared in the same manner as in the Test Example 1.
- The pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector-derived viral solution were produced in the same manner as in the Test Example 1.
- The genes were introduced into the dMEFs by infecting the cells with the retrovirus using the G1NP solution as the viral solution in the same manner as in the Test Example 1.
- An immunostaining analysis was performed using cells 21 days after the introduction to examine expression of insulin, glucagon, somatostatin, and Pdx1.
- Specifically, the cells were fixed with a 4% paraformaldehyde solution at room temperature for 10 min, dipped into a 0.2% Triton-X/PBS solution at room temperature for 10 min, and then treated with 4-fold diluted BLOCKING ONE solution (available from NACALAI TESQUE, INC.) at room temperature for 1 hour. Then, a primary antibody reaction was performed with an anti-insulin antibody (400-fold diluted, guinea pig, available from DAKO), an anti-glucagon antibody (400-fold diluted, rabbit, available from DAKO), an anti-somatostatin antibody (500-fold diluted, rabbit, available from DAKO), or an anti-Pdx1 antibody (1,000-fold diluted, rabbit, obtained from Vanderbilt University, USA) at 4° C. overnight. Then, the resultant reaction products were washed with
PBS 3 times at room temperature for 5 min, and subjected to a secondary antibody reaction with AlexaFluor-Cy3-labeled anti-rabbit antibody (400-fold diluted, available from Invitrogen) or AlexaFluor-488-labeled anti-guinea pig antibody (400-fold diluted, available from Invitrogen) at room temperature for 1 hour. The resultant reaction products were washed withPBS 3 times for 5 min, and then observed and photographed by an inverted fluorescence microscope (CARL ZEISS AXIOVERT 200M). - The results of the immunostaining analysis are presented in
FIGS. 2A to 2C .FIG. 2A illustrates the expression analysis results of insulin and somatostatin;FIG. 2B illustrates the expression analysis results of insulin and glucagon; andFIG. 2C illustrates the expression analysis results of insulin and Pdx1. InFIGS. 2A to 2C , the arrows with a solid line represent cells in which insulin expression is confirmed. InFIGS. 2A to 2C , the arrows with a dashed line represent cells in which somatostatin expression inFIG. 2A , glucagon expression inFIG. 2B , or Pdx1 expression inFIG. 2C is confirmed. - It was confirmed from the results of
FIGS. 2A to 2C that glucagon to be expressed in a cells, insulin to be expressed in β cells, and somatostatin to be expressed in δ cells were expressed at the protein level. Additionally, Pdx1, which is necessary for pancreatic development, was also confirmed to be expressed at the protein level. - Therefore, it was demonstrated that not only β cells, which are insulin producing cells, but also a cells and 8 cells are capable of being produced by the method of the present invention.
- The following cells were prepared.
- (1) dMEF
- Prepared in the same manner as in Test Example 1.
- (2) Human iPS (253G13-6)-derived fibroblasts
- Obtained from Riken BioResource Center.
- (3) Human HepG2 (human hepatoma-derived cell line)
- Obtained from Riken BioResource Center.
- (4) Mouse NIH-3T3 (cultured cells isolated from mouse embryonic skin)
- Obtained from Riken BioResource Center.
- (5) Human embryonic fibroblasts (FHDF)
- Obtained from TOYOBO CO., LTD.
- (6) Human neonatal fibroblasts (NHDF)
- Obtained from TAKARA SHUZO CO., LTD.
- (7) Human HEK293 (cell line established by transforming human embryonic renal cells with Adenovirus E1 gene)
- Obtained from Riken BioResource Center.
- The pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-GFP vector-derived viral solution were produced for mouse cells in the same manner as in the Test Example 1.
- Plat-GP cells in which viral structural proteins gag-pol and env, which were capable of producing high titer viruses for a long period of time, were expressed under the control of MoMuLV LTR and a plasmid DNA (pMX vector or pMX-puro vector, VSVG vector) were used to produce a retrovirus in the following manner (Onishi, M., et. al., Exp. Hematol. 24, 324-329, 1996).
- [pMX-GFP Vector]
- The pMX-GFP vector was prepared in the same manner as in the Test Example 1.
- [pMX-GLIS1 Vector]
- The pMX-GLIS1 vector was prepared in the same manner as in the Test Example 1, except that the sequence of the gene coding for a full-length GLIS1 protein deposited under Accession number NM_147221 was changed to the sequence of the gene coding for a full-length GLIS1 protein deposited under Accession number NM_147193.
- [pMX-Neurogenin3 Vector]
- The pMX-Neurogenin3 vector was prepared in the same manner as in the Test Example 1, except that the sequence of the gene coding for a full-length Neurogenin3 protein deposited under Accession number NM_009719 was changed to the sequence of the gene coding for a full-length Neurogenin3 protein deposited under Accession number NM_020999.
- [pMX-Pdx1 Vector]
- The pMX-Pdx1 vector was prepared in the same manner as in the Test Example 1, except that the sequence of the gene coding for a full-length Pdx1 protein deposited under Accession number NM_008814 was changed to the sequence of the gene coding for a full-length Pdx1 protein deposited under Accession number NM_000209.
- The Plat-GP cells were seeded in a 6-well plate (available from TPP, 92406), which had been coated (for 1 hour at 37° C. and 5% CO2) with Poly-L-Lysine (available from Sigma, P8920) diluted 10 fold with PBS, at 8×105 cells per well, and cultured overnight.
- On the following day, 4 μg of the plasmid DNA (2 μg of the pMX vector and 2 μg of the VSVG vector) was placed into a 1.5 mL tube containing 250 μL of OPTI-MEM (registered trademark) (available from Life Technologies Corporation, 11058021), mixed by tapping, and left to stand at room temperature for 5 min (hereinafter may be referred to as “plasmid/OPTI-MEM solution”). Meanwhile, 10 of LIPOFECTAMINE (registered trademark) 2000 (LP2000) (available from Life Technologies Corporation, 11668500) was placed into another 1.5 mL tube containing 250 μL of OPTI-MEM, mixed together, and left to stand at room temperature for 5 min (hereinafter may be referred to as “LP2000/OPTI-MEM solution”). The plasmid/OPTI-MEM solution and the LP2000/OPTI-MEM solution were well-mixed together and left to stand at room temperature for 20 min (hereinafter may be referred to as “plasmid/LP2000/OPTI-MEM mixed solution”).
- The plasmid/LP2000/OPTI-MEM mixed solution in which liposome-DNA complexes had been formed was added to one well in the 6-well plate, in which the Plat-GP cells seeded the previous day had been cultured, to thereby transfect the cells. After mixing, the cells were cultured within an incubator with 5% CO2 at 37° C. overnight. Twenty-four hours after, the medium was replaced, 1.5 mL of fresh DMEM (containing 10% FBS) was added thereto, and further cultured for 24 hours.
- Forty-eight hours after the transfection, the culture supernatant containing viral particles was collected in a 2.5 mL syringe (available from Terumo Corporation, SS-02SZ) and filtered through a 0.45 filter (available from Whatman, PURADISC FP30 (CA-S 0.45 μm), 10462100) to thereby remove the Plat-GP cells. The culture supernatant containing viral particles were transferred into a 2.0 mL tube.
- Thus, a pMX-GLIS1 vector-derived viral solution, a pMX-Neurogenin3 vector-derived viral solution, a pMX-Pdx1 vector-derived viral solution, and a pMX-GFP vector-derived viral solution for human cells were obtained.
- The genes were introduced into the cells by infecting the cells with the retrovirus using the GIN solution or the GFP CTL solution as the viral solution in the same manner as in the Test Example 1.
- A quantitative PCR analysis was performed as described below using the
cells 20 days after the introduction to thereby determine a relative expression level of an insulin gene relative to a GAPDH gene. - The cells were suspended in a cell lysis solution, and subjected to RNA preparation and cDNA synthesis using SuperPrep™ Cell Lysis & RT Kit for qPCR (available from TOYOBO CO., LTD., #SCQ-101) or SV 96 Total RNA Isolation System (available from Promega, #Z3505), ReverTraAce qPCR RT Master Mix with gDNA Remover (available from TOYOBO CO., LTD., #FSQ-301) and then the quantitative PCR analysis using GeneAce SYBR qPCR Mixα (available from NIPPON GENE CO., LTD.).
- Note that, the following primers were used for the quantitative PCR analysis.
-
-Mouse GAPDH gene- Forward: (SEQ ID NO: 1) 5′-tggagaaacctgccaagtatg-3′ Reverse: (SEQ ID NO: 2) 5′-ggagacaacctggtcctcag-3′ -Mouse insulin2 gene- Forward: (SEQ ID NO: 3) 5′-tttgtcaagcagcacctttg-3′ Reverse: (SEQ ID NO: 4) 5′-ggtctgaaggtcacctgctc-3′ -Human GAPDH gene- Forward: (SEQ ID NO: 5) 5′-atgttcgtcatgggtgtgaa-3′ Reverse: (SEQ ID NO: 6) 5′-tgtggtcatgagtccttcca-3′ -Human insulin gene- Forward: (SEQ ID NO: 7) 5′-gccatcaagcagatcactgt-3′ Reverse: (SEQ ID NO: 8) 5′-caggtgttggttcacaaagg-3′ - The results of the quantitative PCR analysis are presented in
FIGS. 3A to 3G .FIG. 3A illustrates the results of the dMEFs;FIG. 3B illustrates the results of the human iPS (253G13-6)-derived fibroblasts;FIG. 3C illustrates the results of the human HepG2;FIG. 3D illustrates the results of the mouse NIH-3T3;FIG. 3E illustrates the results of the human embryonic fibroblasts;FIG. 3F illustrates the results of the human neonatal fibroblasts; andFIG. 3G illustrates the results of the human HEK293. InFIGS. 3A to 3G , CTL represents the results in the case of using the GFP CTL solution and GIN represents the results in the case of using the GIN solution. Note that, the vertical axis represents the relative expression level of an insulin gene relative to a GAPDH gene. - It was confirmed from the results of
FIGS. 3A to 3G that other cells than mouse fibroblasts also expressed the insulin gene by using the GIN solution. - Therefore, it was demonstrated that pancreatic endocrine cells were capable of being produced from various somatic cells by the method of the present invention.
- The dMEFs were prepared in the same manner as in the Test Example 1.
- [pMX-GFP Vector]
- The pMX-GFP vector was prepared in the same manner as in the Test Example 1.
- [pMX-GLIS1 Vector]
- The pMX-GLIS1 vector was prepared in the same manner as in the Test Example 1.
- [pMX-GLIS3 Vector]
- A pMX-GLIS3 vector is a vector in which a gene coding for a full-length GLIS3 protein is inserted into a multi-cloning site of a pMX vector (obtained from The Institute of Medical Science, The University of Tokyo). Note that, the sequence of the gene coding for a full-length GLIS3 protein is deposited in NCBI under Accession number NM_175459.
- [pMX-Neurogenin3 Vector]
- The pMX-Neurogenin3 vector was prepared in the same manner as in the Test Example 1.
- [pMX-Pdx1 Vector]
- The pMX-Pdx1 vector was prepared in the same manner as in the Test Example 1.
- A culture supernatant containing viral particles was prepared in the same manner as in the Test Example 1, except that the plasmid DNA was used. The resultant culture supernatant was used as a viral solution.
- The genes were introduced into the dMEFs by infecting the cells with the retrovirus in the same manner as in the Test Example 1, except that the following viral solutions were used.
- (1) pMX-GFP vector-derived viral solution (control, hereinafter may be referred to as “GFP CTL solution”);
(2) pMX-GLIS1 vector-derived viral solution (hereinafter may be referred to as “G1 solution”);
(3) pMX-GLIS3 vector-derived viral solution (hereinafter may be referred to as “G3 solution”);
(4) pMX-GLIS1 vector-derived viral solution and pMX-Neurogenin3 vector-derived viral solution (hereinafter may be referred to as “G1N solution”);
(5) pMX-GLIS3 vector-derived viral solution and pMX-Neurogenin3 vector-derived viral solution (hereinafter may be referred to as “G3N solution”);
(6) pMX-GLIS1 vector-derived viral solution, pMX-Neurogenin3 vector-derived viral solution, and pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “G1NP solution”); and
(7) pMX-GLIS3 vector-derived viral solution, pMX-Neurogenin3 vector-derived viral solution, and pMX-Pdx1 vector-derived viral solution (hereinafter may be referred to as “G3NP solution”).
<Counting of Number of dMEF-Derived Insulin Producing Cells> - The number of the DsRed2-positive insulin producing cells was counted in the same manner as in the Test Example 1, except that cells 11 days after the introduction were used.
- The counting results of the DsRed2-positive insulin producing cells are presented in
FIGS. 4A to 4D .FIG. 4A illustrates the results in the case of using the GFP CTL solution (GFP), the G1 solution (G1), and the G1N solution (G1N) as the viral solution from left to right;FIG. 4B illustrates the results in the case of using the GFP CTL solution (GFP), the G1 solution (G1), and the G1NP solution (G1NP) as the viral solution from left to right;FIG. 4C illustrates the results in the case of using the GFP CTL solution (GFP), the G3 solution (G3), and the G3N solution (G3N) as the viral solution from left to right; andFIG. 4D illustrates the results in the case of using the GFP CTL solution (GFP), the G3 solution (G3), and the G3NP solution (G3NP) as the viral solution from left to right. Note that, the vertical axis represents the number of the DsRed2-positive insulin producing cells per well. - It can be seen from the results of
FIGS. 4A to 4D that, even when GLIS3, which was a member of a GUIS family, was used, pancreatic endocrine cells were capable of being produced by introducing along with Neurogenin3, or Neurogenin3 and Pdx1 into somatic cells. Therefore, it was suggested that pancreatic endocrine cells were capable of being produced by introducing the GLIS family along with Neurogenin3, or Neurogenin3 and Pdx1 into somatic cells. It was also demonstrated from comparison of GLIS1 with the GLIS3 that the GLIS1 yields about 10 times higher transdifferentiation efficiency than the GLIS3. - The following cells were prepared.
- (1) Human T98G glioblastoma
- Obtained from Riken BioResource Center.
- (2) Human mesenchymal stem cells
- Obtained from Lonza.
- The pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector- and pMX-GFP vector-derived viral solution were produced in the same manner as Production of retrovirus for human cells in the Test Example 3.
- The genes were introduced into the cells by infecting the cells with the retrovirus using the GFP CTL solution, the G1N solution, or the G1NP solution as the viral solution in the same manner as in the Test Example 1.
- The quantitative PCR analysis was performed in the same manner as in the Test Example 3 to thereby determine a relative expression level of an insulin gene relative to a GAPDH gene.
- The results of the quantitative PCR analysis are presented in
FIGS. 5A and 5B .FIG. 5A illustrates the results of the human T98G glioblastoma; andFIG. 5B illustrates the results of the human mesenchymal stem cells. InFIGS. 5A and 5B , CTL represents the results in the case of using the GFP CTL solution, MN represents the results in the case of using the G1N solution, and G1NP represents the results in the case of using the G1NP solution. Note that, the vertical axis represents the relative expression level of an insulin gene relative to a GAPDH gene. - It was confirmed from the results of
FIGS. 5A and 5B that the insulin gene was expressed also in the human T98G glioblastoma and the human mesenchymal stem cells by using the MN solution or the G1NP solution. - Therefore, it was demonstrated that pancreatic endocrine cells were capable of being produced from various somatic cells by the method of the present invention.
- The dMEFs were prepared in the same manner as in the Test Example 1.
- The pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector-derived viral solution were produced in the same manner as in the Test Example 1.
- The genes were introduced into the dMEFs by infecting the cells with the retrovirus using the G1NP solution as the viral solution in the same manner as in the Test Example 1.
- Cells 16 days after the introduction were observed by a microscopy (FLUO™, available from Leica, AXIOCAM HRC, available from CARL ZEISS, magnification of ×4). Pancreatic islets isolated from mouse pancreas were also observed by the microscopy.
- The results of the microscopic observation are presented in
FIGS. 6A and 6B .FIG. 6A illustrates a pancreatic islet isolated from a mouse pancreas andFIG. 6B illustrates the result in the case of using the G1NP solution as the viral solution. - It was confirmed from the results of
FIGS. 6A and 6B that pancreatic endocrine cell masses obtained by the method of the present invention resemble pancreatic islets isolated from mouse pancreas. - The dMEFs were prepared in the same manner as in the Test Example 1.
- The pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector-derived viral solution were produced in the same manner as in the Test Example 1.
- The genes were introduced into the dMEFs by infecting the cells with the retrovirus using the G1NP solution as the viral solution in the same manner as in the Test Example 1.
- Twenty-seven days after the introduction, 30 uniform pancreatic islet-like masses having a diameter of 100 μm to 300 μm were picked up by a pipette under a stereoscopic microscope and transferred into a 24-well plate. Then, a glucose-responsive insulin secretion test was performed in the same manner.
- The pancreatic islet-like masses were cultured in a 2.8 mM glucose-containing Ringer's solution for 3 hours. Then, the medium was replaced and the masses were cultured for another 1 hour, of which culture supernatants were used as a reference (hereinafter may be referred to as “reference culture supernatant”).
- Then, the pancreatic islet-like masses were cultured in a 16.8 mM glucose-containing Ringer's solution for 1 hour. A culture supernatants thereof were transferred into a 1.5 mL tube (hereinafter may be referred to as “high-glucose culture supernatant”).
- Then, a 2.8 mM glucose-containing Ringer's solution was added to wells, where the pancreatic islet-like masses were cultured for 1 hour. A culture supernatants thereof were transferred into a 1.5 mL tube (hereinafter may be referred to as “low-glucose culture supernatant”).
- An insulin concentration in each of the culture supernatants was measured by ELISA assay (available from Shibayagi Co., Ltd., TYPE T). The results are presented in
FIG. 7 . -
FIG. 7 illustrates the results for each of two wells. InFIG. 7 , (1) illustrates the result of the reference culture supernatant, (2) illustrates the result of the high-glucose culture supernatant, and (3) illustrates the result of the low-glucose culture supernatant. - It was confirmed from the results of
FIG. 7 that an amount of insulin was increased at a higher glucose concentration and a concentration of insulin was decreased at a lower glucose concentration. Therefore, the pancreatic endocrine cells obtained by the method of the present invention were confirmed to have functions required for pancreatic endocrine cells. - Human neonatal fibroblasts (NHDF) (available from TAKARA SHUZO CO., LTD.) were prepared.
- The pMX-GLIS1 vector-derived viral solution, the pMX-Neurogenin3 vector-derived viral solution, and the pMX-Pdx1 vector-derived viral solution were produced in the same manner as in Production of retrovirus for human cells in the Test Example 3.
- The genes were introduced into the human neonatal fibroblasts (NHDF) by infecting the cells with the retrovirus in the same manner as in the Test Example 1, except that the G1NP solution was used as the viral solution and a 24-well plate was used.
- Thirty-four days after the introduction, the entire pancreatic islet-like mass was picked up by a pipette and transferred into a 24-well plate (low attachment plate (EZ-BINDSHUT II, available from IWAKI)). Then, a glucose-responsive insulin secretion test was performed in the following manner.
- The pancreatic islet-like mass was cultured in a 2.8 mM glucose-containing Ringer's solution for 3 hours. Then, the medium was replaced and the mass was cultured for another 1 hour, of which culture supernatant was used as a reference (hereinafter may be referred to as “reference culture supernatant”).
- Then, the pancreatic islet-like mass was cultured in a 25.0 mM glucose-containing Ringer's solution for 1 hour. A culture supernatant thereof was transferred into a 1.5 mL tube (hereinafter may be referred to as “high-glucose culture supernatant”).
- An insulin concentration in each of the culture supernatants was measured by ELISA assay (human insulin ELISA kit, available from Mercodia). The results are presented in
FIG. 8 . - In
FIG. 8 , “Low” represents the result of the reference culture supernatant and “High” represents the result of the high-glucose culture supernatant. - It was confirmed from the results of
FIG. 8 that, also in the case of using human-derived cells, an amount of insulin was increased at a higher glucose concentration and a concentration of insulin was decreased at a lower glucose concentration and that the resultant pancreatic endocrine cells had functions required for pancreatic endocrine cells. - The dMEFs were prepared in the same manner as in the Test Example 1.
- [pCI-GFP Vector]
- A pCI-GFP vector is a vector in which a gene coding for a full-length GFP protein is inserted into a multi-cloning site of a pCI vector (available from Promega) which is an episomal vector. Note that, the sequence of the gene coding for a full-length GFP protein is deposited in NCBI under Accession number L29345.
- [pCI-GLIS1 Vector]
- A pCI-GLIS1 vector is a vector in which a gene coding for a full-length GLIS1 protein is inserted into a multi-cloning site of a pCI vector (available from Promega) which is an episomal vector. Note that, the sequence of the gene coding for a full-length GLIS1 protein is deposited in NCBI under Accession number NM_147221.
- [pCI-Neurogenin3 Vector]
- A pCI-Neurogenin3 vector is a vector in which a gene coding for a full-length Neurogenin3 protein is inserted into a multi-cloning site of a pCI vector (available from Promega) which is an episomal vector. Note that, the sequence of the gene coding for a full-length Neurogenin3 protein is deposited in NCBI under Accession number NM_009719.
- [pCI-Pdx1 Vector]
- A pCI-Pdx1 vector is a vector in which a gene coding for a full-length Pdx1 protein is inserted into a multi-cloning site of a pCI vector (available from Promega) which is an episomal vector. Note that, the sequence of the gene coding for a full-length Pdx1 protein is deposited in NCBI under Accession number NM_008814.
- The following vectors were introduced into the dMEFs by electroporation using NEON (registered trademark) transfection system (available from Life Technologies Corporation). After the vectors were introduced, the cells were incubated within an incubator with 5% CO2 at 37° C. During the incubation, the media (DMEM (containing 10% FBS)) were changed every 2 or 3 days.
- (1) pCI-GFP vector (control, hereinafter may be referred to as “GFP”);
(2) pCI-GLIS1 vector and pCI-Neurogenin3 vector (hereinafter may be referred to as “G1N”); and
(3)pCI-GLIS1 vector, pCI-Neurogenin3 vector, and pCI-Pdx1 vector (hereinafter may be referred to as “G1NP”).
<Counting of Number of dMEF-Derived Insulin Producing Cells> - The number of the DsRed2-positive insulin producing cells was counted in the same manner as in the Test Example 1, except that
cells 8 days after the introduction were used. - The counting results of the DsRed2-positive insulin producing cells are presented in
FIG. 9 . InFIG. 9 , the horizontal axis represents the episomal vectors introduced, that is, the results of the GFP, the G1N, and the G1NP were presented from left to right. Note that, the vertical axis represents the number of DsRed2-positive insulin producing cells per well. - It was demonstrated from the results of
FIG. 9 that, even when the episomal vectors were used, pancreatic endocrine cells were capable of being produced from somatic cells by introducing (I) the GLIS1 gene and the Ngn3 gene or (II) the GLIS1 gene, the Ngn3 gene, and the Pdx1 gene. - A method for producing pancreatic endocrine cells including introducing a gene or one or more gene products thereof into somatic cells according to the present invention is simple, is easily reproduced, and has a remarkably shortened production time compared to previous methods in which pancreatic endocrine cells are produced using ES cells or iPS cells under a culturing environment properly adjusted by, for example, adding a development inhibitor to a medium. According to the method of the present invention, the pancreatic endocrine cells are capable of efficiently produced.
- The method of the present invention is also advantageous in that the pancreatic endocrine cells are capable of being produced without undergoing the iPS cell stage that have a risk of forming tumors.
- Therefore, the method for producing pancreatic endocrine cells according to the present invention is suitably available for, for example, producing pancreatic endocrine cells to be used in regenerative therapies for diabetes.
- Aspects of the present invention are, for example, as follows.
- <1> A method for producing pancreatic endocrine cells, the method including introducing one or more genes of a GLIS family or one or more gene products thereof and a Neurogenin3 gene or one or more gene products thereof into somatic cells.
<2> The method for producing pancreatic endocrine cells according to <1>, wherein the introducing includes further introducing a Pdx1 gene or one or more gene products thereof into the somatic cells.
<3> The method for producing pancreatic endocrine cells according to <1> or <2>, wherein the one or more genes of the GLIS family or one or more gene products thereof are a GLIS1 gene or one or more gene products thereof.
<4> The method for producing pancreatic endocrine cells according to any one of <1> to <3>, wherein the somatic cells are fibroblasts or mesenchymal stem cells.
<5> The method for producing pancreatic endocrine cells according to any one of <1> to <4>, wherein the pancreatic endocrine cells are f3 cells.
<6> Pancreatic endocrine cells produced by the method for producing pancreatic endocrine cells according to any one of <1> to <5>.
<7> The pancreatic endocrine cells according to <6>, wherein the pancreatic endocrine cells include β cells.
<8> A transdifferentiation agent including: - one or more genes of a GLIS family or one or more gene products thereof; and
- a Neurogenin3 gene or one or more gene products thereof,
- wherein the transdifferentiation agent is configured to transdifferentiate somatic cells into pancreatic endocrine cells.
- <9> The transdifferentiation agent according to <8>, further including a Pdx1 gene or one or more gene products thereof.
<10> The transdifferentiation agent according to <8> or <9>, wherein the one or more genes of the GLIS family or one or more gene products thereof is a GLIS1 gene or one or more gene products thereof.
<11> The transdifferentiation agent according to any one of <8> to <10>, wherein the somatic cells are fibroblasts or mesenchymal stem cells.
<12> The transdifferentiation agent according to any one of <8> to <11>, wherein the pancreatic endocrine cells are β cells.
Claims (9)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/883,572 US20220145261A1 (en) | 2014-07-03 | 2020-05-26 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014137719 | 2014-07-03 | ||
JP2014-137719 | 2014-07-03 | ||
PCT/JP2015/069296 WO2016002937A1 (en) | 2014-07-03 | 2015-07-03 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
US201715323576A | 2017-01-03 | 2017-01-03 | |
US16/243,865 US10793832B2 (en) | 2014-07-03 | 2019-01-09 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
US16/883,572 US20220145261A1 (en) | 2014-07-03 | 2020-05-26 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/243,865 Continuation US10793832B2 (en) | 2014-07-03 | 2019-01-09 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
Publications (1)
Publication Number | Publication Date |
---|---|
US20220145261A1 true US20220145261A1 (en) | 2022-05-12 |
Family
ID=55019463
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/323,576 Active US10214728B2 (en) | 2014-07-03 | 2015-07-03 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
US16/243,865 Active US10793832B2 (en) | 2014-07-03 | 2019-01-09 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
US16/883,572 Pending US20220145261A1 (en) | 2014-07-03 | 2020-05-26 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/323,576 Active US10214728B2 (en) | 2014-07-03 | 2015-07-03 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
US16/243,865 Active US10793832B2 (en) | 2014-07-03 | 2019-01-09 | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent |
Country Status (4)
Country | Link |
---|---|
US (3) | US10214728B2 (en) |
EP (1) | EP3165603B1 (en) |
JP (2) | JP6822837B2 (en) |
WO (1) | WO2016002937A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108431212B (en) * | 2015-10-29 | 2022-03-08 | 学校法人顺天堂 | Method for producing pancreatic endocrine cell and transdifferentiator |
WO2019073055A1 (en) | 2017-10-13 | 2019-04-18 | Imba - Institut Für Molekulare Biotechnologie Gmbh | Enhanced reprogramming of somatic cells |
EP4060023A4 (en) | 2019-11-12 | 2023-12-13 | Juntendo Educational Foundation | Method for direct transdifferentiation of somatic cell |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120122214A1 (en) * | 2009-04-24 | 2012-05-17 | National University Corporation Kumamoto University | Method for producing cell medicine |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6911324B2 (en) | 2001-10-18 | 2005-06-28 | The Regents Of The University Of California | Induction of beta cell differentiation in human cells |
JP2003245067A (en) * | 2002-02-25 | 2003-09-02 | Kazutomo Inoue | Intrapancreatic secretory cell |
GB0206357D0 (en) | 2002-03-18 | 2002-05-01 | Univ Bath | Cells |
US20110112015A1 (en) * | 2006-04-14 | 2011-05-12 | Consortum National De Recherche En Genomique (CNRG) | Use of glis3 for preparing functional pancreatic beta-cells |
EP2297298A4 (en) * | 2008-05-09 | 2011-10-05 | Vistagen Therapeutics Inc | Pancreatic endocrine progenitor cells derived from pluripotent stem cells |
US8927277B2 (en) * | 2010-02-16 | 2015-01-06 | Kyoto University | Method of efficiently establishing induced pluripotent stem cells |
-
2015
- 2015-07-03 US US15/323,576 patent/US10214728B2/en active Active
- 2015-07-03 WO PCT/JP2015/069296 patent/WO2016002937A1/en active Application Filing
- 2015-07-03 JP JP2016531476A patent/JP6822837B2/en active Active
- 2015-07-03 EP EP15815940.0A patent/EP3165603B1/en active Active
-
2019
- 2019-01-09 US US16/243,865 patent/US10793832B2/en active Active
-
2020
- 2020-05-26 US US16/883,572 patent/US20220145261A1/en active Pending
- 2020-10-09 JP JP2020171341A patent/JP7072279B2/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120122214A1 (en) * | 2009-04-24 | 2012-05-17 | National University Corporation Kumamoto University | Method for producing cell medicine |
Also Published As
Publication number | Publication date |
---|---|
EP3165603A4 (en) | 2018-01-10 |
US10793832B2 (en) | 2020-10-06 |
JP6822837B2 (en) | 2021-02-03 |
EP3165603B1 (en) | 2019-09-18 |
JPWO2016002937A1 (en) | 2017-04-27 |
US20170211046A1 (en) | 2017-07-27 |
US10214728B2 (en) | 2019-02-26 |
US20190127704A1 (en) | 2019-05-02 |
JP2021035364A (en) | 2021-03-04 |
JP7072279B2 (en) | 2022-05-20 |
WO2016002937A1 (en) | 2016-01-07 |
EP3165603A1 (en) | 2017-05-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6937821B2 (en) | Pluripotent stem cells that can be isolated from living tissues | |
US20220145261A1 (en) | Pancreatic endocrine cells, method for producing same, and transdifferentiation agent | |
Chou et al. | A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach | |
CN103087991A (en) | Method for reprogramming T cells and hematophietic cells | |
Bahrebar et al. | Generation of islet-like cell aggregates from human adipose tissue-derived stem cells by lentiviral overexpression of PDX-1 | |
US20220411762A1 (en) | Method for producing pancreatic endocrine cells, and transdifferentiation agent | |
WO2017159088A1 (en) | Method for preparing cultured cells or cultured tissue for transplantation | |
Chen | Lymphoid Cells | |
JP2019103393A (en) | Method for induction of skeletal muscle stem cell | |
Tobin | Production of lentiviral vectors for gene therapy |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: PUBLICATIONS -- ISSUE FEE PAYMENT VERIFIED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |