Open AccessMature rat hepatocyte dedifferentiation into long lived proliferating hepatic progenitor cells
Author(s) -
А. М. Григорьев,
Irina Kholodenko,
А. Ю. Лупатов,
Р. В. Холоденко,
Л. А. Кирсанова,
Ю. Б. Басок,
К. Н. Ярыгин,
V. I. Sevastianov
Publication year - 2021
Publication title -
vestnik transplantologii i iskusstvennyh organov
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.137
H-Index - 5
eISSN - 2412-6160
pISSN - 1995-1191
DOI - 10.15825/1995-1191-2021-3-148-161
Subject(s) - progenitor cell , hepatocyte , microbiology and biotechnology , wnt signaling pathway , flow cytometry , chemistry , fluorescence microscope , cell , cell growth , progenitor , cell culture , biology , stem cell , signal transduction , in vitro , biochemistry , fluorescence , physics , quantum mechanics , genetics
Objective : to obtain long-lived proliferating cells with progenitor features by dedifferentiation of mature rat hepatocytes using combinations of small molecules. 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 . 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 . Proliferating progenitor cells can be obtained by dedifferentiation of mature hepatocytes.