In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells

Aims: Recently, a new approach to reprogram somatic cells to pluripotent stem cells was shown by fusion of somatic cells with embryonic stem cells (ESC), avoiding generation of totipotent embryos. These fusion hybrids morphologically resemble normal ESCs but have a tetraploid karyotype. Normal hepatocytes often are polyploid, so we preferentially investigate the differentiation potential of fusion hybrids into hepatic cells.

Methods: Mononuclear bone marrow cells from C3H and Rosa26 mice were fused with HM-1 (hypoxanthine-phosphoribosyltransferase -deficient) or OG2 (Oct4-GFP transgenic) ESC, respectively. Unfused ESCs were eliminated by selection with hypoxanthine, aminopterine, thymidine (HAT) for C3H/HM-1 hybrids or G418 for Rosa26/OG2 hybrids and fusion-derived colonies could be subcloned.

Results: Tetraploidy of fusion hybrids was confirmed by FACS analysis of DAPI stained cells and by the presence of the transgenes from both fusion partners in all colonies. The published hepatic precursor differentiation protocols needed slightly modifications. Briefly, pluripotent hybrid cells were cultivated in hanging drops for 5 days before plating onto gelatine-coated dishes for 7 days. Outgrows of these colonies were re-plated on collagen-coated dishes for additional 9 days in Hepatocyte Culture Medium (HCM) to induce hepatic differentiation. Gene expression analyses of these cells showed a hepatic precursor-like expression profile, but weak Oct4-expression still was detectable. In the first set of experiments 3 mice were analyzed after 6 weeks. Transplanted cells show engraftment distributed in all lobes of the liver and no teratoma formation was detectable. But in a second set of experiments 5 out of 8 mice show teratoma formation after 3–5 weeks after transplantation in the spleen (injection site) and the liver.

Conclusions: Murine bone marrow cells are reprogrammed after PEG-mediated fusion with mouse embryonic stem cells and resulting hybrids can be cultured like normal ESCs. A hepatic precursor cell type can be achieved and preliminary transplantation experiments proof engraftment, but functional integration has to be proven and teratoma formation needs to be excluded by introducing selection strategies.