The combination of epidermal growth factor and glycogen synthase kinase 3 inhibitor support long-term self-renewal of Sca-1 positive hepatic progenitor cells from normal adult mice

Cai-Xia Jin, Lisa Samuelson, Cai-Bin Cui, Yang-Zhong Sun, David A. Gerber

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Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, USA.

DOI: 10.4236/scd.2013.33023                

Abstract

Isolation and long-term maintenance of hepatic progenitor cells (HPCs) from healthy, non-injured adult livers remains challenging due to the lack of specific surface markers for selection and a limited understanding of the mechanisms for maintaining self-renewal. Previously, we identified a Sca-1 positive, bipotent HPC population in the peri-portal region of adult liver, and found MAPK/ERK and Wnt/β-Catenin pathways to be synergistically involved in their proliferation. In this study, we report the long-term culture of Sca-1 positive HPCs with epidermal growth factor (EGF) and CHIR99021, a small molecule inhibitor of glycogen synthase kinase 3 (GSK-3). Sca-1+ HPCs remain non-tumorigenic when passaged 35 times in vitro over 1 year. Flow cytometric analysis indicates that HPCs are positive for Sca-1 and putative liver progenitor cell markers, including CD13, CD24 and Prominin-1, but negative for hematopoietic/endothelial cell markers CD31, CD34, CD45, CD90 and CD117. Immunocyto-chemistry and RT-PCR indicate Sca-1+ HPCs express albumin (ALB), α-fetoprotein (AFP), cytokeratin19 (CK19), Sox9 and a panel of special hepatic progenitor transcriptional factors. Moreover, Sca-1+ HPCs are able to differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions in vitro and can take part in liver repopulation in an acetaminophen (APAP) induced liver injury mouse model. This study provides a paradigm to capture and maintain HPCs from naive liver tissue and offers a valuable cell model for investigating the molecular mechanisms underlying the cell lineage relationship in normal liver.

Keywords

Liver Progenitor Cell; Stem Cell Antigen 1; Liver Disease; Hematopoietic Stem Cell

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Azuma, H., Hirose, T., Fujii, H., Oe, S., Yasuchika, K., Fujikawa, T. and Yamaoka, Y. (2003) Enrichment of hepatic progenitor cells from adult mouse liver. Hepatology, 37, 1385-1394. doi:10.1053/jhep.2003.50210
[2]	Fougere-Deschatrette, C., Imaizumi-Scherrer, T., Strick-Marchand, H., Morosan, S., Charneau, P., Kremsdorf, D., Faust, D.M. and Weiss, M.C. (2006) Plasticity of hepatic cell differentiation: Bipotential adult mouse liver clonal cell lines competent to differentiate in vitro and in vivo. Stem Cells, 24, 2098-2109. doi:10.1634/stemcells.2006-0009
[3]	Wang, J., Clark, J.B., Rhee, G.S., Fair, J.H., Reid, L.M. and Gerber, D.A. (2003) Proliferation and hepatic differentiation of adult-derived progenitor cells. Cells Tissues Organs, 173, 193-203. doi:10.1159/000070375
[4]	Wright, N., Samuelson, L., Walkup, M.H., Chandrasekaran, P. and Gerber, D.A. (2008) Enrichment of a bipotent hepatic progenitor cell from naive adult liver tissue. Biochemical and Biophysical Research Communications, 366, 367-372. doi:10.1016/j.bbrc.2007.11.129
[5]	Lazaro, C.A., Croager, E.J., Mitchell, C., Campbell, J.S., Yu, C., Foraker, J., Rhim, J.A., Yeoh, G.C. and Fausto, N. (2003) Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes. Hepatology, 38, 1095-106. doi:10.1053/jhep.2003.50448
[6]	Kamiya, A., Kakinuma, S., Yamazaki, Y. and Nakauchi, H. (2009) Enrichment and clonal culture of progenitor cells during mouse postnatal liver development in mice. Gastroenterology, 137, 1114-1126, 1126 e1-e14.
[7]	Dabeva, M.D., Alpini, G., Hurston, E. and Shafritz, D.A. (1993) Models for hepatic progenitor cell activation. Proceedings of the Society for Experimental Biology and Medicine, 204, 242-252. doi:10.3181/00379727-204-43660
[8]	Wang, X., Foster, M., Al-Dhalimy, M., Lagasse, E., Finegold, M. and Grompe, M. (2003) The origin and liver repopulating capacity of murine oval cells. Proceedings of the National Academy of Sciences of USA, 100, 11881-11888. doi:10.1073/pnas.1734199100
[9]	Theise, N.D., Saxena, R., Portmann, B.C., Thung, S.N., Yee, H., Chiriboga, L., Kumar, A. and Crawford, J.M. (1999) The canals of Hering and hepatic stem cells in humans. Hepatology, 30, 1425-1433. doi:10.1002/hep.510300614
[10]	Jelnes, P., Santoni-Rugiu, E., Rasmussen, M., Friis, S.L., Nielsen, J.H., Tygstrup, N. and Bisgaard, H.C. (2007) Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration. Hepatology, 45, 1462-1470. doi:10.1002/hep.21569
[11]	Jin, C., Samuelson, L., Cui, C.B., Sun, Y. and Gerber, D.A. (2011) MAPK/ERK and Wnt/beta-Catenin pathways are synergistically involved in proliferation of Sca-1 positive hepatic progenitor cells. Biochemical and Biophysical Research Communications, 409, 803-807. doi:10.1016/j.bbrc.2011.05.094
[12]	Ring, D.B., Johnson, K.W., Henriksen, E.J., Nuss, J.M., Goff, D., Kinnick, T.R., Ma, S.T., Reeder, J.W., Samuels, I., Slabiak, T., Wagman, A.S., Hammond, M.E. and Harrison, S.D. (2003) Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo. Diabetes, 52, 588-595. doi:10.2337/diabetes.52.3.588
[13]	Seglen, P.O. (1976) Preparation of isolated rat liver cells. Methods in Cell Biology, 13, 29-83. doi:10.1016/S0091-679X(08)61797-5
[14]	O’Brien, L.E., Yu, W., Tang, K., Jou, T.S., Zegers, M.M. and Mostov, K.E. (2006) Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures. Methods in Enzymology, 406, 676-691.
[15]	Furuyama, K., Kawaguchi, Y., Akiyama, H., Horiguchi, M., Kodama, S., Kuhara, T., Hosokawa, S., Elbahrawy, A., Soeda, T., Koizumi, M., Masui, T., Kawaguchi, M., Takaori, K., Doi, R., Nishi, E., Kakinoki, R., Deng, J.M., Behringer, R.R., Nakamura, T. and Uemoto, S. (2011) Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nature Genetics, 43, 34-41. doi:10.1038/ng.722
[16]	Nierhoff, D., Ogawa, A., Oertel, M., Chen, Y.Q. and Shafritz, D.A. (2005) Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity. Hepatology, 42, 130-139. doi:10.1002/hep.20735
[17]	Quintana-Bustamante, O., Alvarez-Barrientos, A., Kofman, A.V., Fabregat, I., Bueren, J.A., Theise, N.D. and Segovia, J.C. (2006) Hematopoietic mobilization in mice increases the presence of bone marrow-derived hepatocytes via in vivo cell fusion. Hepatology, 43, 108-116. doi:10.1002/hep.21005
[18]	Fukuda, A., Kawaguchi, Y., Furuyama, K., Kodama, S., Horiguchi, M., Kuhara, T., Koizumi, M., Boyer, D.F., Fujimoto, K., Doi, R., Kageyama, R., Wright, C.V. and Chiba, T. (2006) Ectopic pancreas formation in Hes1-knockout mice reveals plasticity of endodermal progenitors of the gut, bile duct, and pancreas. Journal of Clinical Investigation, 116, 1484-1493. doi:10.1172/JCI27704