Therapeutic effect of MSCs against liver injury In a model of drug-induced chronic liver disease, a previous report has shown that transplanted MSCs can be involved in anti- fibrotic effects. The authors also showed that transplanted MSCs scattered mostly in the hepatic connective tissue and survived in the liver 4 weeks after transplantation, but did not differentiate into hepatocytes expressing albumin or alpha-fetoprotein [5]. This report suggested that a variety of bioactive cytokines secreted by the transplanted MSCs might be involved in restoring liver function and promoting regeneration. In addition, another report has shown that MSC-conditioned medium has the potential to dramatically reduce cell death [12,13]. On the other hand, certain studies have described the transdifferentiation of MSCs into cells with a hepatocyte-like phenotype [14,15,16,17]. Models of chronic liver disease have a relatively long time for the onset of cell therapy effects to be established, and the period of observation is also relatively longer after the MSCs are transplanted. It is still controversial whether transplanted MSCs protect and regenerate the liver by cell fusion or by transdifferentiation, or by neither. Thus, further studies on the fate of MSCs after transplantation are necessary. In models of acute liver disease due to I/R injury, free radicals generated during the acute phase of I/R injury initiate the inflammatory cascade, giving rise to the second attack, which is characterized by infiltration of activated neutrophils in the liver promptly after reperfusion. Activation of Kupffer cells and T lymphocytes promotes neutrophil recruitment, assisted by increased endothelial expression of adhesion molecules. There- fore, transplanted MSCs need to work efficiently shortly after reperfusion. In the present study, MSC treatment ameliorated the increase in serum transaminase levels, which serves as the most sensitive marker for clinical and experimental hepatic I/R injury evaluation. These findings indicate that MSCs were viable and able to function shortly after transplantation. There is limited time for MSCs to transdifferentiate into hepatocytes or hepatocyte-like cells during the acute phase. Therefore, it is unlikely that transdifferentiation is involved in tissue protection and repair. Histopathological findings showed that hepatocyte apoptosis induced by I/R injury mainly existed in the centrilobular region in the Control group, whereas LacZ-positive MSCs were detected in the periportal area in the MSC group. Transplanted MSCs did not replace damaged hepatocytes, but settled down in the periportal area. This evidence supports the notion that paracrine actions exerted by MSCs through the release of soluble factors might be important for tissue protection and repair. Moreover, the luciferase activity of MSCs gradually decreased and at 168 h after reperfusion, we could not find any LacZ-positive cells in the liver sections (data not shown). Once the liver fully recovers, it is possible that host hepatocytes identify the transplanted MSCs as non-self cells and eliminate them from the liver. Seventy-two hours after reperfusion, a degree of liver injury was improved in the MSC group compared to the Control group. It seems reasonable that MSC transplantation attenuating I/R injury would result in the histopathological differences observed between the MSC group and the Control group. Also, the sequence of events might affect liver regeneration and cause a significant difference in remnant liver regeneration 168 h after reperfusion. In conclusion, these findings suggest that MSCs might have the potential to protect the liver against I/R injury-induced hepato- cyte apoptosis, and to enhance liver regeneration. Impact of MSCs against Hepatic I/R Injury Materials and Methods Animal All experiments were conducted under the approval of the Jichi Medical University Guide for Laboratory Animals (Approval number: # 1080). Male wild Lewis rats were purchased from Charles River (Breeding Laboratories, Kanagawa, Japan). Rats used in the experiments had a body weight of between 230 and 310 g. The animals were housed in a temperature- and humidity- controlled environment with a 12 h light/12 h dark cycle with free access to food (standard laboratory chow) and water ad libitum. After fasting overnight, all animals were anesthetized with ether inhalation. Establishment of double transgenic rats Double transgenic (Tg) rats expressing luciferase and LacZ were created by crossbreeding ROSA/luciferase Tg Lewis rats [18] with ROSA/LacZ Lewis rats [19]. IVIS was used to detect luciferase expression, and X-gal staining was used to detect LacZ expression (detailed below). The F1 hybrids between ROSA/ luciferase Tg and ROSA/LacZ Lewis rats were imaged after intravenous injection of D-luciferin (30 mg/kg/body weight) (potassium salt; Bio-synth, Postfach, Switzerland), followed by X- gal staining. In the same manner, luciferase and LacZ expression levels were examined in various tissues of the rats. Approximately one-fourth of the F1 hybrids expressed luciferase and LacZ in the whole body. We used these ‘‘dual colored’’ F1 hybrids expressing both luciferase and LacZ (luc/LacZ) as our MSC donors. Bone marrow-derived MSC preparation and culture Bone marrow cells were isolated from female double (luc/LacZ) Tg rat femurs by flushing the femurs with aMEM (invitorgen, Tokyo) supplemented with 10% fetal bovine saline (invitrogen, Tokyo) and antibiotic-antimycotic (invitrogen, Tokyo), using a 19- gauge needle. Isolated bone marrow cells were seeded onto 10-ml tissue culture dishes (Thermo Scientific, Tokyo), and cultured with aMEM supplemented with 10% FBS. When the cells were 70%– 80% confluent, they were harvested with 0.05% trypsin-EDTA (invitrogen, Tokyo), replated at 26104 cells/cm2, and cultured for 5 days. MSCs between the fifth and eighth passage were used for the experiments. Operative procedure A temporary warm ischemia of the liver was induced as shown in Figure 8. All surgical procedures were performed under light ether anesthesia. The abdomen was opened through a midline incision and ligaments surrounding each lobe were then dissected away. Clamping the portal vein, the hepatic artery, and the bile duct supplying the median and the left lateral lobes of the liver with a microvessel clip induced 70% partial liver ischemia. This technique enabled us to avoid intestinal congestion, which may lead to fatal hemodynamic instability [20,21]. After 40 min of partial hepatic ischemia, the vessel clip was released to initiate hepatic reperfusion. Immediately after onset of reperfusion, the nonischemic lobes (the superior right lateral, the inferior right lateral, the anterior caudate and the posterior caudate lobes) were excised. Furthermore, these procedures were followed by left lateral and left portion of the medial lobectomy, leaving only the ischemic right portion of medial lobe behind. Subsequently, phosphate-buffered saline (PBS) or 16106 MSCs in a volume of 200 ml were transfused into the portal vein with a 30-gauge needle for over 1 min. The abdomen was closed with 3-0 silk sutures, and PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e19195