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EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Scalable Selection of Hepatocyte- and Hepatocyte Precursor-Like Cells from Culture of Differentiating Transgenically Modified Murine Embryonic Stem Cells

Irina Drobinskaya^a, Thomas Linn^b, Tomo ari^a, Reinhard G. Bretzel^b, Heribert Bohlen^c, Jürgen Hescheler^a, Eugen Kolossov^c

^aInstitute of Neurophysiology, Center of Physiology and Pathophysiology, University of Cologne, Cologne, Germany;
^bMedical Clinic and Policlinic III, Justus-Liebig University of Giessen and Marburg, Giessen, Germany;
^c Axiogenesis AG, Cologne, Germany

Key Words. Murine embryonic stem cells • Transgenic cell clones • Hepatocyte-like cells • In vitro differentiation • Antibiotic selection

Correspondence: Correspondence: Irina Drobinskaya, Ph.D., Institute for Neurophysiology, Center of Physiology and Pathophysiology, University of Cologne, Robert-Koch Str. 39, D-50931 Cologne, Germany. Telephone: + 49-221-478-86772; Fax: + 49-221-478-3834; e-mail: irina.drobinskaya{at}uni-koeln.de; or Eugen Kolossov, Ph.D., Axiogenesis AG, Nattermannallee 1, Building S20, D-50829 Cologne, Germany. Telephone: + 49-221-99-881818; Fax: + 49-221-99-881810; e-mail: eugen.kolossov{at}axiogenesis.com

Received on April 22, 2008; accepted for publication on June 2, 2008.

First published online in STEM CELLS EXPRESS  June 12, 2008.

Potential therapeutic applications of embryonic stem cell (ESC)-derived hepatocytes are limited by their relatively low output in differentiating ESC cultures, as well as by the danger of contamination with tumorigenic undifferentiated ESCs. To address these problems, we developed transgenic murine ESC clones possessing bicistronic expression vector that contains the -fetoprotein gene promoter driving a cassette for the enhanced green "live" fluorescent reporter protein (eGFP) and a puromycin resistance gene. Under established culture conditions these clones allowed for both monitoring of differentiation and for puromycin selection of hepatocyte-committed cells in a suspension mass culture of transgenic ESC aggregates ("embryoid bodies" [EBs]). When plated on fibronectin, the selected eGFP-positive cells formed colonies, in which intensely proliferating hepatocyte precursor-like cells gave rise to morphologically differentiated cells expressing -1-antitrypsin, -fetoprotein, and albumin. A number of cells synthesized glycogen and in some of the cells cytokeratin 18 microfilaments were detected. Major hepatocyte marker genes were expressed in the culture, along with the gene and protein expression of stem/progenitor markers, suggesting the features of both hepatocyte precursors and more advanced differentiated cells. When cultured in suspension, the EB-derived puromycin-selected cells formed spheroids capable of outgrowing on an adhesive substrate, resembling the behavior of fetal mouse hepatic progenitor cells. The established system based on the highly efficient selection/purification procedure could be suitable for scalable generation of ESC-derived hepatocyte- and hepatocyte precursor-like cells and offers a potential in vitro source of cells for transplantation therapy of liver diseases, tissue engineering, and drug and toxicology screening.

Disclosure of potential conflicts of interest is found at the end of this article.