We modified the silver staining method of Kerenyi and Gallyas (Clin Chim Acta 1972; 38: 465-467) for visualizing low levels of protein with our own agarose gels as reported previously (Yokomizo K. et al. J Electrophoresis 2003; 47: 91-97). To reduce background staining and avoid the formation of artifact spots, a number of factors were studied. These included the composition of the staining reagent and fixative solutions (first and second fixation). It was found that 2.5% Na₂CO₃, 0.1% AgNO₃, 0.1% NH₄NO₃, 0.75% tungtosilicic acid and 0.14% formaldehyde of the staining reagent mixture was critical and that the addition of 5% ZnSO₄ to the second fixative solution resulted in consistently lower background staining and a significant reduction in artifact spots. Our silver staining procedure is approximately 100-fold more sensitive than Coomassie brilliant blue and the detection limit of bovine serum albumin is about 1.46 ng per band.

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The transplanted GFP-positive BMCs (especially the Liv8 negative cell population, without culturing) migrated into the peri-portal regions of the cirrhotic liver. They differentiated into Liv2-positive hepatoblasts and then into albumin-producing hepatocytes. The differentiation “niche” induced by persistent liver damage due to continuous CCl₄ injection seems to be an essential factor. Microarry-SOM analysis showed that at an early stage after BMC transplantation, the genes related to morphology were activated. Then later, genes associated with liver metabolism were activated. Finally, BMC transplantation improved liver function, liver fibrosis and the survival rate. These findings strongly support the development of a new cell therapy using autologous BMCs to treat liver cirrhosis patients, because BMC transplantation itself is an established treatment for hematological diseases. Our finding indicates that FGF2 will accelerate the differentiation of BMC to hepatocyte.
Based on the results obtained in basic research using the GFP/CCl₄ model, human trials are now undergoing.

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