Figure 2 Hepatocyte preconditioning of hBM‐MSC in vitro. (A) Morphological changes. During differentiation, hBM‐MSCs change from a fibroblast‐like spindle shape (undifferentiated) to a hepatocyte‐like polygonal shape as culture proceeds (day 7 and day 15). (B) Detection of hepatocyte‐specific proteins. Immunocytochemical staining (upper panels) and detection by Western blot (bottom panels) of CK18, CX32, HepPar1 and PCK1 in cells before differentiation (u) and after different days of culture in differentiation medium (day 21 (upper) and days 4, 11, 31 (bottom), respectively). (C) Expression of hepatocyte‐specific RNA transcripts. Total RNA was extracted from hBM‐MSCs before differentiation (u) and after different times of culture in differentiation medium (days 7, 15, 22). RNA transcripts typically expressed in early‐ (AFP, CK19, CK7, CX43) and late‐ (CYP3A4, ALB, CPS, CK18, CX32, PCK1, TFN) differentiated hepatocytes were detected by RT–PCR after normalisation to the amount of GAPDH transcripts as an internal standard (not shown). WB, Western blot.