RU2016100433A - КЛЕТКИ SC-β И КОМПОЗИЦИИ И СПОСОБЫ ДЛЯ ИХ СОЗДАНИЯ - Google Patents
КЛЕТКИ SC-β И КОМПОЗИЦИИ И СПОСОБЫ ДЛЯ ИХ СОЗДАНИЯ Download PDFInfo
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- RU2016100433A RU2016100433A RU2016100433A RU2016100433A RU2016100433A RU 2016100433 A RU2016100433 A RU 2016100433A RU 2016100433 A RU2016100433 A RU 2016100433A RU 2016100433 A RU2016100433 A RU 2016100433A RU 2016100433 A RU2016100433 A RU 2016100433A
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- cell
- glucose
- cells
- pancreatic
- stimulation
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- 238000000034 method Methods 0.000 title claims 16
- 239000000203 mixture Substances 0.000 title claims 10
- 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 claims 28
- 239000008103 glucose Substances 0.000 claims 28
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 claims 20
- 230000000638 stimulation Effects 0.000 claims 18
- 210000004027 cell Anatomy 0.000 claims 17
- 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 claims 12
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- 102000051325 Glucagon Human genes 0.000 claims 4
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- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical class IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 claims 4
- 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 claims 4
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- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims 3
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- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims 3
- 108090000695 Cytokines Proteins 0.000 claims 2
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- 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 claims 2
- 229960000553 somatostatin Drugs 0.000 claims 2
- 239000005495 thyroid hormone Substances 0.000 claims 2
- 229940036555 thyroid hormone Drugs 0.000 claims 2
- 229940035722 triiodothyronine Drugs 0.000 claims 2
- VOUAQYXWVJDEQY-QENPJCQMSA-N 33017-11-7 Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)CCC1 VOUAQYXWVJDEQY-QENPJCQMSA-N 0.000 claims 1
- 102100024645 ATP-binding cassette sub-family C member 8 Human genes 0.000 claims 1
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- 102100027886 Homeobox protein Nkx-2.2 Human genes 0.000 claims 1
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- 101000760570 Homo sapiens ATP-binding cassette sub-family C member 8 Proteins 0.000 claims 1
- 101000614701 Homo sapiens ATP-sensitive inward rectifier potassium channel 11 Proteins 0.000 claims 1
- 101000873786 Homo sapiens Glutamate decarboxylase 2 Proteins 0.000 claims 1
- 101000632186 Homo sapiens Homeobox protein Nkx-2.2 Proteins 0.000 claims 1
- 101000578254 Homo sapiens Homeobox protein Nkx-6.1 Proteins 0.000 claims 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 claims 1
- 101000971533 Homo sapiens Killer cell lectin-like receptor subfamily G member 1 Proteins 0.000 claims 1
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- 101000613495 Homo sapiens Paired box protein Pax-4 Proteins 0.000 claims 1
- 101000591210 Homo sapiens Receptor-type tyrosine-protein phosphatase-like N Proteins 0.000 claims 1
- 101000979205 Homo sapiens Transcription factor MafA Proteins 0.000 claims 1
- 101000802379 Homo sapiens Zinc transporter 10 Proteins 0.000 claims 1
- 101000818846 Homo sapiens Zinc transporter 8 Proteins 0.000 claims 1
- 102100023915 Insulin Human genes 0.000 claims 1
- 102000017792 KCNJ11 Human genes 0.000 claims 1
- 102100021457 Killer cell lectin-like receptor subfamily G member 1 Human genes 0.000 claims 1
- 102100028192 Mitogen-activated protein kinase kinase kinase kinase 2 Human genes 0.000 claims 1
- 101710144533 Mitogen-activated protein kinase kinase kinase kinase 2 Proteins 0.000 claims 1
- 101150079937 NEUROD1 gene Proteins 0.000 claims 1
- 108700020297 NeuroD Proteins 0.000 claims 1
- 102100032132 Neuroendocrine convertase 1 Human genes 0.000 claims 1
- 102100032063 Neurogenic differentiation factor 1 Human genes 0.000 claims 1
- 108010032788 PAX6 Transcription Factor Proteins 0.000 claims 1
- 102100040909 Paired box protein Pax-4 Human genes 0.000 claims 1
- 102100037506 Paired box protein Pax-6 Human genes 0.000 claims 1
- 102100022308 Ras-related protein Rab-3A Human genes 0.000 claims 1
- 102100034091 Receptor-type tyrosine-protein phosphatase-like N Human genes 0.000 claims 1
- 108091006296 SLC2A1 Proteins 0.000 claims 1
- 108010057722 Synaptosomal-Associated Protein 25 Proteins 0.000 claims 1
- 102000004183 Synaptosomal-Associated Protein 25 Human genes 0.000 claims 1
- 102100021417 Zinc transporter 8 Human genes 0.000 claims 1
- 239000000853 adhesive Substances 0.000 claims 1
- 239000003472 antidiabetic agent Substances 0.000 claims 1
- 229940125708 antidiabetic agent Drugs 0.000 claims 1
- 230000006907 apoptotic process Effects 0.000 claims 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 claims 1
- 238000012258 culturing Methods 0.000 claims 1
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- 239000008187 granular material Substances 0.000 claims 1
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- OGQSCIYDJSNCMY-UHFFFAOYSA-H iron(3+);methyl-dioxido-oxo-$l^{5}-arsane Chemical compound [Fe+3].[Fe+3].C[As]([O-])([O-])=O.C[As]([O-])([O-])=O.C[As]([O-])([O-])=O OGQSCIYDJSNCMY-UHFFFAOYSA-H 0.000 claims 1
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Claims (37)
1. Композиция, содержащая ненативную панкреатическую β-клетку, при этом:
(a) ненативная панкреатическая β-клетка содержит одну или более кристаллических инсулиновых гранул;
(b) ненативная панкреатическая β-клетка экспрессирует следующие гены: INS, PDX1, NKX6-1 и ZNT8; и
(c) ненативная панкреатическая β-клетка демонстрирует in vitro ответ, состоящий в стимулированной глюкозой секреции инсулина, на первую стимуляцию глюкозой.
2. Композиция по п. 1, отличающаяся тем, что ненативная панкреатическая β-клетка демонстрирует in vitro ответ, состоящий в стимулированной глюкозой секреции инсулина, на первую стимуляцию глюкозой, вторую стимуляцию глюкозой и третью стимуляцию глюкозой, при этом первую стимуляцию глюкозой, вторую стимуляцию глюкозой и третью стимуляцию глюкозой проводят последовательно.
3. Композиция по п. 1, отличающаяся тем, что ненативная панкреатическая β-клетка дополнительно экспрессирует по меньшей мере один ген, выбранный их группы, состоящей из MAFA, РАХ6, NEUROD1, GCK, SLC2A1, PCSK1, KCNJ11, АВСС8, SNAP25, RAB3A, GAD2, PTPRN, NKX2-2 и РАХ4.
4. Композиция по п. 1, отличающаяся тем, что ненативная панкреатическая β-клетка секретирует инсулин в ответ на первую концентрацию глюкозы по сравнению со второй концентрацией глюкозы в соотношении, составляющем по меньшей мере 1,1, при этом первая концентрация глюкозы выше, чем вторая концентрация глюкозы.
5. Композиция по п. 1, отличающаяся тем, что ненативная панкреатическая β-клетка является моногормональной.
6. Композиция по п. 1, отличающаяся тем, что количество инсулина, секретируемого ненативной панкреатической β-клеткой, составляет по меньшей мере 0,5 мкМЕ на 1000 клеток за 30 минут инкубации, при этом ненативную панкреатическую β-клетку обрабатывают по меньшей мере 20 мМ глюкозы.
7. Композиция по п. 1, отличающаяся тем, что секреция инсулина из ненативной панкреатической β-клетки повышается в ответ на противодиабетический агент.
8. Композиция по п. 1, отличающаяся тем, что ненативная панкреатическая β-клетка демонстрирует цитокин-индуцированный апоптоз в ответ на цитокин.
9. Композиция по п. 1, отличающаяся тем, что ненативная панкреатическая β-клетка характеризуется профилем генной экспрессии, который отличается от профиля генной экспрессии нативной β-клетки.
10. Популяция клеток, содержащая одну или более ненативных панкреатических β-клеток по п. 1, отличающаяся тем, что по меньшей мере 10% клеток в популяции клеток являются ненативными панкреатическими β-клетками.
11. Популяция клеток по п. 12, дополнительно содержащая инсулин-позитивные эндокринные клетки.
12. Популяция клеток по п. 12, отличающаяся тем, что популяция клеток содержит одну из следующих клеток:
a. С-пептид-негативную/глюкагон-позитивную клетку;
b. С-пептид-негативную/соматостатин-позитивную клетку;
c. глюкагон-позитивную/соматостатин-негативную клетку;
d. глюкагон-негативную/соматостатин-позитивную клетку; или
e. любую их комбинацию.
13. Популяция клеток по п. 12, отличающаяся тем, что по меньшей мере 3% популяции клеток составляют С-пептид-негативные/глюкагон-позитивные клетки или глюкагон-позитивные/соматостатин-негативные клетки.
14. Искусственные островок или поджелудочная железа, содержащие популяцию клеток по п. 12.
15. Способ дифференцировки клеток-предшественников в панкреатические β-клетки in vitro, включающий культивирование кластера клеток-предшественников в суспензии в культуральной среде, содержащей активатор сигнального пути тиреоидного гормона и ингибитор сигнального пути трансформирующего фактора роста β (TGF-β), вследствие чего происходит дифференцировка клеток-предшественников в панкреатические β-клетки, при этом панкреатические β-клетки демонстрируют in vitro ответ, состоящий в стимулированной глюкозой секреции инсулина, по меньшей мере на первую стимуляцию глюкозой.
16. Способ по п. 15, отличающийся тем, что клетки-предшественники включают инсулин-позитивную эндокринную клетку.
17. Способ по п. 15, отличающийся тем, что клетки-предшественники включают PDX1 -позитивную клетку-предшественник.
18. Способ по п. 15, отличающийся тем, что активатором сигнального пути тиреоидного гормона является трииодотиронин.
19. Способ по п. 18, отличающийся тем, что концентрация трииодотиронина составляет по меньшей мере 0,1 мМ.
20. Способ по п. 15, отличающийся тем, что ингибитором сигнального пути TGF-β является ингибитор II Alk5.
21. Способ по п. 20, отличающийся тем, что концентрация ингибитора II Alk5 составляет по меньшей мере 100 нМ.
22. Способ по п. 20, отличающийся тем, что концентрация ингибитора II Alk5 составляет 100 мкМ или меньше.
23. Способ по п. 15, отличающийся тем, что культивирование включает замену культуральной среды по меньшей мере через день.
24. Способ по п. 15, отличающийся тем, что культивирование длится по меньшей мере семь дней.
25. Способ по п. 15, отличающийся тем, что культуральная среда является бессывороточной культуральной средой.
26. Способ по п. 15, отличающийся тем, что клетки-предшественники культивируют в присутствии низкоадгезивного субстрата.
27. Способ по п. 15, отличающийся тем, что клетки-предшественники являются человеческими клетками-предшественниками.
28. Способ по п. 15, отличающийся тем, что панкреатические β-клетки демонстрируют in vitro ответ, состоящий в стимулированной глюкозой секреции инсулина, на первую стимуляцию глюкозой и вторую стимуляцию глюкозой, при этом первую стимуляцию глюкозой и вторую стимуляцию глюкозой проводят последовательно.
29. Способ по п. 15, отличающийся тем, что панкреатические β-клетки демонстрируют in vitro ответ, состоящий в стимулированной глюкозой секреции инсулина, на первую стимуляцию глюкозой, вторую стимуляцию глюкозой и третью стимуляцию глюкозой, при этом первую стимуляцию глюкозой, вторую стимуляцию глюкозой и третью стимуляцию глюкозой проводят последовательно.
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