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RU2016121409A - SUSPENDING AND CLUSTERING OF HUMAN PLURIPOTENT STEM CELLS WITH THE PURPOSE OF THEIR DIFFERENTIATION IN PANCREATIC ENDOCRINE CELLS - Google Patents

SUSPENDING AND CLUSTERING OF HUMAN PLURIPOTENT STEM CELLS WITH THE PURPOSE OF THEIR DIFFERENTIATION IN PANCREATIC ENDOCRINE CELLS Download PDF

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RU2016121409A
RU2016121409A RU2016121409A RU2016121409A RU2016121409A RU 2016121409 A RU2016121409 A RU 2016121409A RU 2016121409 A RU2016121409 A RU 2016121409A RU 2016121409 A RU2016121409 A RU 2016121409A RU 2016121409 A RU2016121409 A RU 2016121409A
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cells
pluripotent
clusters
medium
differentiating
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RU2016121409A
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RU2689710C2 (en
RU2016121409A3 (en
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Бенджамин ФРАЙЕР
Даина ЛАНИАУСКАС
Марсия БЛЭКМУР
Хайюнь ВАН
Костадинка ЛИЛОВА
Шелли НЕЛЬСОН
Элизабет РОСОЧА
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Янссен Байотек, Инк.
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Claims (26)

1. Способ выращивания и дифференцировки плюрипотентных клеток в динамически перемешиваемой суспензионной культуральной системе, включающий культивирование плюрипотентных клеток в плоской прикрепленной культуре до агрегированных кластеров клеток и дифференцировку кластеров плюрипотентных клеток в динамической перемешиваемой суспензионной культуральной системе, в котором стадия дифференцировки включает использование ингибитора Cyp26.1. A method of growing and differentiating pluripotent cells in a dynamically agitated suspension culture system, comprising cultivating pluripotent cells in a flat attached culture to aggregated cell clusters and differentiating clusters of pluripotent cells in a dynamically agitated suspension culture system, wherein the differentiation step involves the use of a Cyp26 inhibitor. 2. Способ по п.1, причем указанный способ увеличивает процент клеток в фазе G0/G1 клеточного цикла.2. The method according to claim 1, wherein said method increases the percentage of cells in the G0 / G1 phase of the cell cycle. 3. Способ по п.1, в котором культивирование включает среду, содержащую от приблизительно 0,1% до приблизительно 2% бычьего сывороточного альбумина.3. The method according to claim 1, in which the cultivation includes a medium containing from about 0.1% to about 2% bovine serum albumin. 4. Способ по п.3, в котором среда дополнительно включает в себя ингибитор Rho-киназы.4. The method according to claim 3, in which the medium further includes an Rho kinase inhibitor. 5. Способ по п.1, в котором плюрипотентные клетки являются прикрепленными.5. The method according to claim 1, in which the pluripotent cells are attached. 6. Способ увеличения процента клеток в фазе G0/G1 клеточного цикла, включающий выращивание плюрипотентных клеток в плоской прикрепленной культуре до агрегированных кластеров клеток в среде, включающей в себя от приблизительно 0,1% до приблизительно 2% бычьего сывороточного альбумина, перенос кластеров плюрипотентных стволовых клеток из плоской прикрепленной культуры в динамическую суспензию и культивирование клеток в динамической суспензии в среде с добавлением малой молекулы и, необязательно, представителя семейства TGFβ.6. A method for increasing the percentage of cells in the G0 / G1 phase of the cell cycle, comprising growing pluripotent cells in a flat attached culture to aggregated cell clusters in a medium comprising from about 0.1% to about 2% bovine serum albumin, transferring pluripotent stem clusters cells from a flat attached culture into a dynamic suspension and culturing the cells in a dynamic suspension in a medium with the addition of a small molecule and, optionally, a member of the TGFβ family. 7. Способ по п.6, в котором малая молекула представляет собой MCX, и причем представитель семейства TGFβ представляет собой GDF-8.7. The method according to claim 6, in which the small molecule is an MCX, and wherein the representative of the TGFβ family is GDF-8. 8. Способ по п.6, в котором выращивание включает среду, содержащую ингибитор Rho-киназы.8. The method according to claim 6, in which the cultivation includes a medium containing an Rho kinase inhibitor. 9. Способ дифференцировки плюрипотентных клеток в дефинитивную энтодерму, включающий выращивание плюрипотентных клеток в плоской прикрепленной культуре до агрегированных кластеров клеток в среде, включающей в себя от приблизительно 0,1% до приблизительно 2% бычьего сывороточного альбумина, перенос кластеров плюрипотентных стволовых клеток из плоской прикрепленной культуры в динамическую суспензию и культивирование клеток в динамической суспензии в среде с добавлением MCX и GDF8 или WNT3A и активина A.9. A method for differentiating pluripotent cells into a definitive endoderm, comprising growing pluripotent cells in a flat attached culture to aggregated cell clusters in a medium comprising from about 0.1% to about 2% bovine serum albumin, transferring pluripotent stem cell clusters from a flat attached culture in a dynamic suspension and culturing cells in a dynamic suspension in a medium supplemented with MCX and GDF8 or WNT3A and activin A. 10. Способ по п.9, в котором выращивание включает среду, содержащую ингибитор Rho-киназы.10. The method according to claim 9, in which the cultivation includes a medium containing an Rho kinase inhibitor. 11. Способ выращивания и дифференцировки плюрипотентных стволовых клеток в динамически перемешиваемой суспензионной культуральной системе, включающий выращивание плюрипотентных стволовых клеток в плоской прикрепленной культуре до агрегированных кластеров клеток, перенос кластеров плюрипотентных стволовых клеток из плоской прикрепленной культуры в динамическую суспензию и дифференцировку плюрипотентных клеток в динамически перемешиваемой суспензионной культуральной системе, причем клетки выращивают до агрегированных кластеров клеток в среде, которая включает в себя от приблизительно 0,1% до приблизительно 2% бычьего сывороточного альбумина.11. A method of growing and differentiating pluripotent stem cells in a dynamically stirred suspension culture system, comprising growing pluripotent stem cells in a flat attached culture to aggregated cell clusters, transferring clusters of pluripotent stem cells from a flat attached culture to a dynamic suspension and differentiating pluripotent cells in a dynamically mixed suspension the culture system, and the cells are grown to aggregate clusters cells in a medium that includes from about 0.1% to about 2% bovine serum albumin. 12. Способ по п.11, в котором кластеры плюрипотентных клеток дифференцируют для создания популяции панкреатических клеток-предшественников, популяции нейронных клеток-предшественников или популяции клеток-предшественников кардиомиоцитов.12. The method of claim 11, wherein the pluripotent cell clusters are differentiated to create a population of pancreatic progenitor cells, a population of neural progenitor cells, or a population of cardiomyocyte progenitor cells. 13. Способ по п.11, в котором плюрипотентные стволовые клетки выбираются из следующих: индуцированные плюрипотентные стволовые клетки, клетки, полученные из ткани пуповины человека, партеноты, человеческие эмбриональные стволовые клетки (клетки hES) и клетки, извлеченные из амниотической жидкости.13. The method according to claim 11, in which the pluripotent stem cells are selected from the following: induced pluripotent stem cells, cells derived from human umbilical cord tissue, parthenotes, human embryonic stem cells (hES cells), and cells extracted from amniotic fluid. 14. Трансплантируемый клеточный продукт, полученный из стволовых клеток, содержащий популяцию дифференцированных панкреатических клеток-предшественников, полученных посредством способа по п.11.14. A transplanted cell product derived from stem cells containing a population of differentiated pancreatic progenitor cells obtained by the method of claim 11. 15. Способ выращивания и дифференцировки плюрипотентных клеток в динамически перемешиваемой суспензионной культуральной системе, включающий:15. A method of growing and differentiating pluripotent cells in a dynamically stirred suspension culture system, including: a. выращивание плюрипотентных клеток в плоской прикрепленной культуре до агрегированных кластеров клеток в среде, включающей в себя от приблизительно 0,1% до приблизительно 2% бычьего сывороточного альбумина;a. growing pluripotent cells in a flat attached culture to aggregated clusters of cells in a medium comprising from about 0.1% to about 2% bovine serum albumin; b. перенос кластеров плюрипотентных клеток с плоской прикрепленной культуры в динамическую суспензионную культуру с использованием фермента или хелатирующего агента;b. the transfer of clusters of pluripotent cells from a flat attached culture in a dynamic suspension culture using an enzyme or chelating agent; c. поддержание кластеров клеток в динамически перемешиваемой суспензионной культуральной системе; иc. maintaining cell clusters in a dynamically stirred suspension culture system; and d. дифференцировку кластеров плюрипотентных клеток в динамически перемешиваемой суспензионной культуральной системе для генерирования популяции панкреатических клеток-предшественников, популяции нейронных клеток-предшественников или популяции клеток-предшественников кардиомиоцитов;d. differentiating clusters of pluripotent cells in a dynamically stirred suspension culture system to generate a population of pancreatic progenitor cells, a population of neural progenitor cells, or a population of cardiomyocyte progenitor cells; причем дифференцировочная среда включает в себя кислород в диапазоне от недостаточности кислорода до приблизительно 30% от уровня окружающей среды, уровень липидов в диапазоне от 0,1% до приблизительно 2% или их комбинацию.moreover, the differentiating medium includes oxygen in the range from oxygen deficiency to about 30% of the ambient level, lipid levels in the range from 0.1% to about 2%, or a combination thereof. 16. Способ по п.15, в котором кластеры стволовых клеток экспрессируют CD9, SSEA4, TRA-1-60 и TRA-1-81, и не экспрессируют CXCR4.16. The method of claim 15, wherein the stem cell clusters express CD9, SSEA4, TRA-1-60, and TRA-1-81, and do not express CXCR4. 17. Способ по п.15, в котором среда для выращивания дополнительно включает ингибитор Rho-киназы.17. The method of claim 15, wherein the growth medium further comprises an Rho kinase inhibitor. 18. Способ по п.15, в котором среда для дифференцировки содержит от приблизительно 0,1% до приблизительно 2% бычьего сывороточного альбумина.18. The method according to clause 15, in which the medium for differentiation contains from about 0.1% to about 2% bovine serum albumin. 19. Способ по п.15, в котором плюрипотентные клетки выбирают из группы, состоящей из индуцированных плюрипотентных стволовых клеток, клеток, полученных из ткани пуповины человека, партенотов, человеческих эмбриональных стволовых клеток (клетки hES) и клеток, извлеченных из амниотической жидкости.19. The method of claim 15, wherein the pluripotent cells are selected from the group consisting of induced pluripotent stem cells, cells derived from human umbilical cord tissue, parthenotes, human embryonic stem cells (hES cells), and cells extracted from amniotic fluid. 20. Способ по п.15, в котором по данному способу генерируются панкреатические клетки-предшественники, экспрессирующие фактор транскрипции β-клеток.20. The method according to clause 15, in which the method generates pancreatic progenitor cells expressing the transcription factor of β-cells. 21. Способ по п.15, в котором факторами транскрипции являются PDX1 и/или NKX6.1. 21. The method according to clause 15, in which the transcription factors are PDX1 and / or NKX6.1.
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