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RU2009140903A - Способ перепрограммирования соматических клеток - Google Patents

Способ перепрограммирования соматических клеток Download PDF

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Publication number
RU2009140903A
RU2009140903A RU2009140903/10A RU2009140903A RU2009140903A RU 2009140903 A RU2009140903 A RU 2009140903A RU 2009140903/10 A RU2009140903/10 A RU 2009140903/10A RU 2009140903 A RU2009140903 A RU 2009140903A RU 2009140903 A RU2009140903 A RU 2009140903A
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Prior art keywords
cells
cell
somatic
purified
reprogramming
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RU2009140903/10A
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English (en)
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RU2502799C2 (ru
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Рудольф ДЖЭНИШ (US)
Рудольф ДЖЭНИШ
Джейкоб ХАННА (US)
Джейкоб ХАННА
Мариус ВЕРНИГ (US)
Мариус ВЕРНИГ
Кристофер Дж. ЛЕНГНЕР (US)
Кристофер Дж. ЛЕНГНЕР
Александр МАЙССНЕР (US)
Александр МАЙССНЕР
Оливер Тобиас БРЭМБРИНК (US)
Оливер Тобиас БРЭМБРИНК
Рут ФОРМАН (US)
Рут ФОРМАН
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Уайтхед Инститьют Фор Биомедикал Рисёч (Us)
Уайтхед Инститьют Фор Биомедикал Рисёч
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Publication of RU2009140903A publication Critical patent/RU2009140903A/ru
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Publication of RU2502799C2 publication Critical patent/RU2502799C2/ru

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Abstract

1. Очищенный препарат выделенных плюрипотентных клеток млекопитающего, отличающийся тем, что клетки (а) экспрессируют эндогенные Oct4 и Nanog; (б) будучи инъецированы SCID мышам, дифференцируются в клетки тканей, имеющих характеристики эндодермы, мезодермы и эктодермы; (в) не экспрессируют введенный экзогенно ген плюрипотентности или селективный маркер, функционально связанный с эндогенным геном плюрипотентности, (г) являются перепрограммированными соматическими клетками млекопитающего. ! 2. Очищенный препарат клеток по п.1, отличающийся тем, что по меньшей мере 50% клеток резистентны к деметилированию ДНК. ! 3. Очищенный препарат клеток по п.1, отличающийся тем, что клетки выживают в условиях, в которых экспрессия ДНК-метилтрансферазы I снижается по меньшей мере на 50%. ! 4. Очищенный препарат клеток по п.1, отличающийся тем, что клетки не являются генетически модифицированными. ! 5. Очищенный препарат клеток по п.1, отличающийся тем, что клетки содержат по меньшей мере одну генную модификацию. ! 6. Очищенный препарат клеток по п.1, отличающийся тем, что клетки генетически соответствуют донору указанных соматических клеток или донору клетки-предшественника указанных соматических клеток, причем указанный донор является индивидуумом, нуждающимся в клеточной терапии. ! 7. Способ генерации перепрограммированной соматической клетки, включающий использование соматической клетки, которая содержит один или более введенный экзогенно ген, который содействует перепрограммированию указанной клетки в плюрипотентное состояние, причем указанная клетка перепрограммируется в плюрипотентное состояние, в котором клетки (i) резистентны �

Claims (25)

1. Очищенный препарат выделенных плюрипотентных клеток млекопитающего, отличающийся тем, что клетки (а) экспрессируют эндогенные Oct4 и Nanog; (б) будучи инъецированы SCID мышам, дифференцируются в клетки тканей, имеющих характеристики эндодермы, мезодермы и эктодермы; (в) не экспрессируют введенный экзогенно ген плюрипотентности или селективный маркер, функционально связанный с эндогенным геном плюрипотентности, (г) являются перепрограммированными соматическими клетками млекопитающего.
2. Очищенный препарат клеток по п.1, отличающийся тем, что по меньшей мере 50% клеток резистентны к деметилированию ДНК.
3. Очищенный препарат клеток по п.1, отличающийся тем, что клетки выживают в условиях, в которых экспрессия ДНК-метилтрансферазы I снижается по меньшей мере на 50%.
4. Очищенный препарат клеток по п.1, отличающийся тем, что клетки не являются генетически модифицированными.
5. Очищенный препарат клеток по п.1, отличающийся тем, что клетки содержат по меньшей мере одну генную модификацию.
6. Очищенный препарат клеток по п.1, отличающийся тем, что клетки генетически соответствуют донору указанных соматических клеток или донору клетки-предшественника указанных соматических клеток, причем указанный донор является индивидуумом, нуждающимся в клеточной терапии.
7. Способ генерации перепрограммированной соматической клетки, включающий использование соматической клетки, которая содержит один или более введенный экзогенно ген, который содействует перепрограммированию указанной клетки в плюрипотентное состояние, причем указанная клетка перепрограммируется в плюрипотентное состояние, в котором клетки (i) резистентны к деметилированию ДНК; (ii) экспрессируют эндогенный Oct4; и (iii) будучи инъецированы SCID мышам, дифференцируются в клетки тканей, имеющих характеристики эндодермы, мезодермы и эктодермы.
8. Способ по п.7, отличающийся тем, что два или более введенных экзогенно генов выбирают из группы, содержащей Oct-4, Nanog, Sox-2, c-Myc, Lin28 и Klf4.
9. Способ по п.8, отличающийся тем, что два или более введенных экзогенно генов представляют собой Oct-4 и Nanog.
10. Способ по п.8, отличающийся тем, что два или более введенных экзогенно генов представляют собой Oct-4 и Sox-2.
11. Способ по п.8, отличающийся тем, что два или более введенных экзогенно генов представляют собой Oct-4 и Klf-4.
12. Способ по п.7, отличающийся тем, что один или более введенных экзогенно генов выбирают из группы, содержащей Oct-4, Nanog, Sox-2, c-Myc, Klf4 и их комбинации.
13. Способ по п.7, отличающийся тем, что клетка содержит введенные экзогенно гены, кодирующие Oct-4, Nanog, Sox-2, c- Myc и Klf4 или сами белки.
14. Способ по п.7, отличающийся тем, что один или более введенных экзогенно генов вводят путем трансфекции.
15. Способ по п.7, отличающийся тем, что один или более введенных экзогенно генов вводят путем вирусного инфицирования.
16. Способ по п.7, дополнительно включающий функциональную инактивацию по меньшей мере одного из указанных введенных экзогенно генов.
17. Способ по п.16, отличающийся тем, что включает эксцизию по меньшей мере участка указанного введенного экзогенно гена с помощью введения в указанную клетку сайт-специфической рекомбиназы или экспрессии сайт-специфической рекомбиназы в клетке.
18. Способ перепрограммирования дифференцированной соматической клетки в плюрипотентное состояние, включающий стадии:
(а) контактирование дифференцированной соматической клетки по меньшей мере с одним перепрограммирующим агентом, который способствует перепрограммированию указанной клетки в плюрипотентное состояние; и
(б) сохранение указанной клетки в условиях, подходящих для пролиферации указанной клетки и для активности указанного по меньшей мере одного перепрограммирующего агента в течение периода времени, достаточного для того, чтобы начать перепрограммирование указанной клетки.
19. Способ по п.18, отличающийся тем, что указанный по меньшей мере один перепрограммирующий агент представляет собой полинуклеотид.
20. Способ по п.18, отличающийся тем, что указанный по меньшей мере один перепрограммирующий агент представляет собой полипептид.
21. Способ по п.19 или 20, отличающийся тем, что один или более перепрограммирующий агент выбирают из группы, содержащей Oct-4, Sox-2, Klf4, Nanog, Lin28, c-Myc и их комбинаций.
22. Способ по п.18, отличающийся тем, что перепрограммирующий агент выбирают из группы, содержащей 5-азацитидин, трихостатин A (TSA) и вальпроевую кислоту.
23. Способ по п.18, отличающийся тем, что дифференцированная соматическая клетка представляет собой частично дифференцированную клетку.
24. Способ по п.18, отличающийся тем, что дифференцированная соматическая клетка представляет собой полностью дифференцированную клетку.
25. Способ идентификации агента, который перепрограммирует соматические клетки в менее дифференцированное состояние, включающий:
(а) контактирование соматических клеток с предполагаемым перепрограммирующим агентом, причем соматические клетки чувствительны к пониженному ДНК-метилированию; и
(б) определение количества клеток, резистентных к пониженному ДНК-метилированию, причем возрастание числа клеток, резистентных к пониженному ДНК-метилированию, по сравнению с контролем указывает, что предполагаемый агент является перепрограммирующим агентом.
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