High-purity hepatic lineage differentiated from dental pulp stem cells in serum-free medium.

INTRODUCTION: We have previously differentiated hepatocyte like cells from deciduous tooth pulp stem and extracted third molar pulp stem cells with a protocol that used fetal bovine serum, but it showed high contaminations of nondifferentiated cells. Both the lower purity of hepatically differentiated cells and usage of serum are obstacles for application of cell therapy or regenerative medicine. Objective of this study was to investigate the capacity for and purity of hepatocyte-like differentiation of CD117-positive dental pulp stem cells without serum. METHODS: Mesenchymal cells from human deciduous and extracted third molar pulp were isolated and expanded in vitro. We separated CD117-positive cells by using a magnetic-activated cell sorter. The cells were characterized immunofluorescently by using known stem cell markers. For hepatic differentiation, the media were supplemented with hepatic growth factor, insulin-transferrin-selenium-x, dexamethasone, and oncostatin M. Expression of hepatic markers alpha fetoprotein, albumin, hepatic nuclear factor-4 alpha, insulin-like growth factor-1, and carbamoyl phosphate synthetase was examined immunofluorescently after differentiation. The amount of differentiated cells was assessed by using flow cytometry. Glycogen storage and urea concentration in the medium were defined. RESULTS: Both cell cultures demonstrated a number of cells positive for all tested hepatic markers after differentiation, ie, albumin-positive cells were almost 90% of differentiated deciduous pulp cells. The concentration of urea in the media increased significantly after differentiation. Significant amount of cytoplasmic glycogen storage was found in the cells. CONCLUSIONS: Without serum both cell types differentiated into high-purity hepatocyte-like cells. These cells offer a source for hepatocyte lineage differentiation for transplantation in the future.

Mitochondrial steps of arginine biosynthesis are conserved in the hydrogenosomes of the chytridiomycete Neocallimastix frontalis.

Arginine biosynthesis in eukaryotes is divided between the mitochondria and the cytosol. The anaerobic chytridiomycete Neocallimastix frontalis contains highly reduced, anaerobic modifications of mitochondria, the hydrogenosomes. Hydrogenosomes also occur in the microaerophilic flagellate Trichomonas vaginalis, which does not produce arginine but uses one of the mitochondrial enzymes, ornithine transcarbamoylase, in a cytosolic arginine dihydrolase pathway for ATP generation. EST sequencing and analysis of the hydrogenosomal proteome of N. frontalis provided evidence for two mitochondrial enzymes of arginine biosynthesis, carbamoylphosphate synthase and ornithine transcarbamoylase, while activities of the arginine dehydrolase pathway enzymes were not detectable in this fungus.