Analysis of protein expression profile by DIGE A proteomic DIGE approach was used to analyze the repertoire of proteins differentially expressed in control cells and hepatocytes obtained with CM1 or CM2 differentiation protocols. The DIGE analysis showed 39 differentially expressed proteins, and 17 of them were identified, including done chaperones, metabolic, structural, proteolytic and apoptosis-related proteins (Table 2). Eleven of these proteins were differentially expressed in CM1 vs. CM2 (Figure 6). The differential expression in CM1 vs. CM2 of proteins, such as adenine phosphoribosyl transferase, transgelin, cathepsine B precursor, tropomyosin �� chain and L-lactate dehydrogenase �� chain was confirmed by western blots (Figure 5B).

DIGE analysis showed a higher expression of adenine phosphoribosyltransferase, cathepsin B and D, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or L-lactate dehydrogenase ��-chain in hepatocytes obtained after treatment with CM2, than in CM1-treated or undifferentiated cells. In contrast, the expression of other proteins, such as transgelin, tropomyosin �� chain, annexin A5 or Dna J homologous subfamily B decreased in hepatocytes obtained after treatment with CM2, compared to CM1-treated or undifferentiated cells. Nuclear ��-catenin was also more expressed after treatment with CM2 than in CM1-treated cells. Figure 6 The activation of Wnt/��-catenin during hepatocyte differentiation is associated with the presence of related proteins to tumoral phenotype.

Table 2 Comparative analysis by DIGE of proteins differentially expressed in hepatocytes obtained with CM1 or CM2 differentiation protocols. Discussion Hepatocytes differentiation has been achieved using different types of stem cells, MSC [17], embryonic stem cells [18] or induced pluripotent stem cells [19]. However in these studies the role of Wnt/��-catenin activation during hepatogenesis is unclear. In our study, we used human MSC Batimastat and two different protocols to achieve differentiation into hepatocytes; one without Wnt/��-catenin activation (CM1) and other with Wnt signaling activation (CM2). The expression of hepatospecific genes and the key regulator of hepatogenesis CEBP were achieved in both protocols. Similar differentiation results has been obtained by others authors using other stem cells [20]. Wnt/��-catenin pathway activation took place in CM2-treated cells, with nuclear ��-catenin translocation and up-regulation of genes related to this pathway. Treatment of cells with another protocol (CM1) also induced hepatic differentiation but without the concurrence activation of Wnt/��-catenin pathway. We show for the first time the capability of CM1 (HGF+FGF7) to differentiate human MSC into hepatocytes.