vtioВестник трансплантологии и искусственных органовRussian Journal of Transplantology and Artificial Organs1995-1191Academician V.I.Shumakov National Medical Research Center of Transplantology and Artificial Organs", Ministry of Health of the Russian Federation10.15825/1995-1191-2021-3-148-161vtio-1383Research ArticleРЕГЕНЕРАТИВНАЯ МЕДИЦИНА И КЛЕТОЧНЫЕ ТЕХНОЛОГИИREGENERATIVE MEDICINE AND CELL TECHNOLOGIESДедифференцировка зрелых крысиных гепатоцитов в длительно пролиферирующие печеночные прогениторные клетки.Mature rat hepatocyte dedifferentiation into long lived proliferating hepatic progenitor cellsГригорьевА. М.GrigorievA. M.

Григорьев Алексей Михайлович

123182, Москва, ул. Щукинская, д. 1

 

Alexey M. Grigoryev

1, Shchukinskaya str., Moscow, 123182

Bear-38@yandex.ruХолоденкоИ. В.KholodenkoI. V.

Холоденко Ирина Викторовна

Москва

Moscow

irkhol@yandex.ruЛупатовА. Ю.LupatovA. Y.

Лупатов Алексей Юрьевич

Москва

Moscow

alupatov@mail.ruХолоденкоР. В.KholodenkoR. V.

Холоденко Роман Васильевич

Москва

Moscow

khol@mail.ruКирсановаЛ. А.KirsanovaL. A.

Кирсанова Людмила Анфилофьевна

Москва

Moscow

lyudochkakirsanova@mail.ruБасокЮ. Б.BasokY. B.

Басок Юлия Борисовна

Москва

Moscow

bjb2005@mail.ruЯрыгинК. Н.YaryginK. N.

Ярыгин Константин Никитич

Москва

Moscow

kyarygin@yandex.ruСевастьяновВ. И.SevastianovV. I.

Севастьянов Виктор Иванович

Москва

Moscow

viksev@yandex.ruФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава РоссииРоссияShumakov National Medical Research Center of Transplantology and Artificial OrgansRussian FederationФГБНУ «Научно-исследовательский институт биомедицинской химии имени В.Н. Ореховича»РоссияOrekhovich Institute of Biomedical ChemistryRussian FederationФГБУН «Институт биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова»РоссияShemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Federation202117092021233148161Copyright © Григорьев А.М., Холоденко И.В., Лупатов А.Ю., Холоденко Р.В., Кирсанова Л.А., Басок Ю.Б., Ярыгин К.Н., Севастьянов В.И., 20212021Григорьев А.М., Холоденко И.В., Лупатов А.Ю., Холоденко Р.В., Кирсанова Л.А., Басок Ю.Б., Ярыгин К.Н., Севастьянов В.И.Grigoriev A.M., Kholodenko I.V., Lupatov A.Y., Kholodenko R.V., Kirsanova L.A., Basok Y.B., Yarygin K.N., Sevastianov V.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.transpl.ru/vtio/article/view/1383Цель

Цель: получить длительно пролиферирующие клетки, обладающие признаками прогениторных, за счет дедифференцировки зрелых крысиных гепатоцитов с помощью комбинаций малых молекул.

Материалы и методы

Материалы и методы. Гепатоциты, выделенные из крысиной печени путем перфузии, культивировали в присутствии набора из трех малых молекул: агониста Wnt сигнального пути (CHIR99021), ингибиторов TGF-β (A83-01) и ROCK киназы (Y27632). С помощью флуоресцентной и фазово-контрастной микроскопии в процессе культивирования клеток оценивали морфологические характеристики и особенности роста культуры. Пролиферативную активность клеток анализировали с помощью цейтраферной съемки в режиме реального времени. Экспрессию поверхностных и внутриклеточных маркеров анализировали, используя проточную цитофлуориметрию и флуоресцентную микроскопию высокого разрешения.

Результаты

Результаты. Используя комплекс малых молекул Y-27632, A-83-01 и CHIR99021, из зрелых крысиных гепатоцитов были получены длительно пролиферирующие клетки, которые экспрессировали маркеры прогениторных клеток, такие как α-фетопротеин и HNF4α. Клетки имели гепатоцитоподобную морфологию и формировали дискретные кластеры пролиферирующих клеток, образующих в процессе культивирования единый клеточный пласт. Удаление из среды малых молекул приводило к экспансии фибробластоподобных клеток и к элиминации потенциально прогениторных гепатоцитоподобных клеток.

Заключение

Заключение. Подтверждена возможность получения пролиферирующих прогениторных клеток с помощью дедифференцировки зрелых гепатоцитов.

Objective

Objective: to obtain long-lived proliferating cells with progenitor features by dedifferentiation of mature rat hepatocytes using combinations of small molecules.

Materials and Methods

Materials and Methods. Hepatocytes isolated from rat liver by perfusion were cultured in the presence of a cocktail of three small molecules – Wnt signaling pathway activator (CHIR99021), TGF-β inhibitors (A83-01) and ROCK kinase (Y27632). The morphological characteristics and growth features of the culture were assessed using fluorescence and phase-contrast microscopy during cell culture. Cell proliferative activity was analyzed using real-time time-lapse imaging. The expression of surface and intracellular markers was analyzed using flow cytometry and high-resolution fluorescence microscopy.

Results

Results. Using a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, long-lived proliferating cells that express progenitor cell markers, such as α-fetoprotein and HNF4α, were obtained from mature rat hepatocytes. The cells had hepatocyte-like morphology and formed discrete clusters of proliferating cells, forming a single cell layer during culturing. Removal of the small molecules from the medium led to expansion of fibroblast-like cells and elimination of potentially progenitor hepatocyte-like cells.

Conclusion

Conclusion. Proliferating progenitor cells can be obtained by dedifferentiation of mature hepatocytes.

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The authors declare that there are no conflicts of interest present.