An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.
Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.
Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.
Ayaka
A
Yanagida
Keiichi
K
Ito
Hiromi
H
Chikada
Hiromitsu
H
Nakauchi
Akihide
A
Kamiya
D000071916:AC133 Antigen; D000577:Amides / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D018826:CD13 Antigens / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D016207:Cytokines / Q000494:pharmacology; D061252:Feeder Cells / Q000166:cytology; D005434:Flow Cytometry; D006023:Glycoproteins / Q000378:metabolism; D051557:Hepatocyte Nuclear Factor 4 / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D053552:Keratin-7 / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013859:Thiocarbamates / Q000494:pharmacology; D013882:Thiosemicarbazones; D000509:alpha-Fetoproteins / Q000378:metabolism
C507011:A-83-01; D000071916:AC133 Antigen; D000577:Amides; D015703:Antigens, CD; D015415:Biomarkers; D016207:Cytokines; D006023:Glycoproteins; D051557:Hepatocyte Nuclear Factor 4; D036341:Intercellular Signaling Peptides and Proteins; D053552:Keratin-7; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; D011720:Pyrazoles; D011725:Pyridines; D013859:Thiocarbamates; D013882:Thiosemicarbazones; D000509:alpha-Fetoproteins; C108830:Y 27632; D003577:Cytochrome P-450 Enzyme System; D018826:CD13 Antigens