Results Characterization of BM-MSCs Bone marrow-derived mesenchymal stem cells (BM-MSCs) were spindle shaped and plastic-adherent cells in standard culture conditions (Figure 1A). BM-MSCs were characterized by immunofluorescence. We identified the expression of CD29+, CD105+, CD312, and CD342 on BM-MSCs by immunostaining (Figure 1B, 1C, 1D, 1E). Previous data showed that rat MSCs were positive for CD29 and CD105, but were negative for CD31 and CD34 [7,8]. Differentiation ability of BM-MSCs into classical mesenchymal lineage cells including adipocytes, osteo- blasts, or chondrocytes was verified by using previously reported methods (Figure 2). The results indicated that the cells were undifferentiated and had stem cell characteristics. Effect of BM-MSCs on increased levels of serum ALT induced by I/R in rats Serum AST and ALT levels were measured to determine damage to hepatocytes 24 h after reperfusion. Serum ALT levels were significantly decreased in the MSC group com- pared with the Control group (MSC group: 11366325 IU/L, Control group: 21986854 IU/L, P,0.03). Serum AST levels were also decreased (MSC group: 10436223 IU/L, Control group: 13776428 IU/L, but not significantly). However, liver sections of the MSC group were not morphologically dis- Impact of MSCs against Hepatic I/R Injury tinguishable (H&E) from sections of the Control group (data not shown). Decreased luciferase activity in MSC rats Twenty-four hours after reperfusion, most of the MSCs were detected in the remnant liver by In Vivo Imaging System (IVIS), and thereafter, luciferase activity diminished with time (Figure 3). When luciferase was measured 168 h after reperfusion, the amount of light emitted was only slightly detectable. BM-MSC transplantation inhibits apoptosis after I/R injury TUNEL-positive hepatocytes in the Control group were mainly localized in the centrilobular region (Figure 4A). The extent of hepatocyte apoptosis was evaluated by TUNEL staining 6 h after reperfusion. TUNEL staining yielded a mean of 10.560.7 positively stained cells/high power field in the Control group, and 0.560.5 in the MSC group (P,0.03) (Figure 4C). X-gal immunostaining reveals localization of BM-MSCs in the liver Almost all of the LacZ-positive BM-MSCs were distributed around the portal triad and interlobular connective tissue 6 h after reperfusion (Figure 5A). There were very few LacZ-positive BM- MSCs in the centrilobular region. When the periportal area was examined more closely, BM-MSCs were also detected in the sinusoids (Figure 5B). Figure 1. Characterization of BM-MSCs by expression of CD29+, CD312, CD342, and CD105+. A. Bright-field image. B–C. CD29 and CD105 surface antigens are positive. D. CD31 surfice antigen is negative, E. CD34 surfice antigen negative. doi:10.1371/journal.pone.0019195.g001 PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e19195