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Long-Term Culture and Differentiation of Rat Embryonic Stem Cell-Like Cells into Neuronal, Glial, Endothelial, and Hepatic Lineages

^a Department of General and Thoracic Surgery, University Hospital of Kiel, Kiel, Germany;
^b Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany

Key Words. Rat embryonic stem cells • In vitro differentiation • Long-term culture • Hepatic lineage • Fibroblast growth factor-4

Maren Ruhnke, M.D., Department of General and Thoracic Surgery, University Hospital of Kiel, Arnold-Heller Str.7, 24105 Kiel, Germany. Telephone: 49-431-5974482; Fax: 49-431-5971995; e-mail: marenschulze1{at}aol.com

The in vitro differentiation of mouse embryonic stem cells into different somatic cell types such as neurons, endothelial cells, or myocytes is a well-established procedure. Long-term culture of rat embryonic stem cells is known to be hazardous, and attempts to differentiate these cells in vitro so far have been unsuccessful. We herein describe stable long-term culture of an alkaline phosphatase-positive rat embryonic stem cell-like cell line (RESC) and its differentiation into neuronal, endothelial, and hepatic lineages. RESCs were characterized by typical growth in single cells as well as in embryoid bodies when cultured in the presence of leukemia inhibitory factor. RESC expressed stage-specific-embryonic antigen-1 and the major histocompatibility complex class I molecule. For neuronal differentiation, cells were incubated with medium containing 10^-6 M retinoic acid for 14 days. For endothelial differentiation, RESCs were grown on Matrigel for 14 days, and for induction of hepatocyte-specific antigen expression, RESCs were grown in medium supplemented with fibroblast growth factor-4. Differentiated cells exhibited typical morphological changes and expressed neuronal (nestin, mitogen-activated protein-2, synaptophysin), glial (S100, glial fibrillary acid protein), endothelial (panendothelial antibody, CD31) and hepatocyte-specific (-fetoprotein [FP], albumin, -1-antitrypsin, CK18) antigens. In addition, expression of hepatocyte-specific genes (FP, transthyretin, carbamoyl-phosphate synthetase, and coagulation factor-2) was detected by reverse transcription polymerase chain reaction. We were able to culture RESCs under stable, long-term conditions and to initiate programmed differentiation of RESCs to endothelial, neuronal, glial, and hepatic lineages in the rat species.

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