Fig 1.
Experimental protocol for canine model of liver fibrosis and treatment.
The canines were administered repeated carbon tetrachloride (CCl4) injections through the implanted catheter for 10 weeks (high-dose period: 1.0 mL/kg body weight once per week and 0.5 mL/kg body weight once per week for 6 weeks; low-dose period: 0.25 mL/kg body weight twice per week for 4 weeks). CCl4 was injected on the first and fourth days of each week. At 0 weeks (0W; i.e. after 10 weeks of CCl4 injections), the canines were divided into three groups. In the control group, low-dose CCl4 injection was continued for a further 12 weeks without bone marrow-derived mesenchymal stem cell (BMSC) infusion. In the Vein group, cultured autologous canine BMSCs (4 × 105/kg) were infused through a peripheral vein, and low-dose CCl4 injections were continued for 12 more weeks. In the Artery group, cultured autologous BMSCs (4 × 105/kg) were infused through a hepatic artery using angiography, and low-dose CCl4 injections were continued for 12 more weeks. In all three groups, blood assays, indocyanine green (ICG) testing, and liver biopsy based on ultrasonography were performed at −10W, 0W, 4W, 8W, and 12W, as indicated by the arrows.
Fig 2.
Ventral-dorsal digital subtraction images of a canine.
Hepatic arteriogram was performed after the injection of contrast medium through a 4-French catheter placed in the common hepatic artery. We inserted a catheter in the left hepatic artery, right medial lobe branch, and right lateral lobe branch and injected bone marrow-derived mesenchymal stem cells (BMSCs) via each of these arteries at doses of 2 × 105 cell/kg, 1 × 105 cell/kg, and 1 × 105 cell/kg, respectively.
Fig 3.
Liver fibrosis in different treatment groups as assessed by Sirius-red staining.
The canines were administered repeated carbon tetrachloride (CCl4) injections to induce liver fibrosis and treated with bone marrow-derived mesenchymal stem cells (BMSCs) via the peripheral vein (Vein group) or hepatic artery (Artery group). (a) In the Control group, the fibrosis level increased over time from 11.3 ± 3.9% (0W) to 12.4 ± 4.3% (4W), 12.7 ± 2.6% (8W), and 12.9 ± 2.8% (12W). (b) In the Vein group, the fibrosis level decreased from 9.2 ± 2.7% (0W) to 7.2± 3.5% (4W), 6.4 ± 2.7% (8W), and 6.9 ± 2.7% (12W). (c) In the Artery group, the fibrosis level decreased significantly from 11.0 ± 2.5% (0W) to 7.2 ± 1.2% (4W; p < 0.05), 6.8 ± 1.3% (8W; p < 0.05), and 6.9 ± 1.0% (12W; p < 0.05). 4W, 8W, and 12W denote weeks post-BMSC administration.
Fig 4.
Quantification of liver fibrosis assessed by Sirius red staining in different treatment groups.
The canines were administered repeated carbon tetrachloride (CCl4) injections to induce liver fibrosis and treated with bone marrow-derived mesenchymal stem cells (BMSCs) via the peripheral vein (Vein group) or hepatic artery (Artery group). (a) Significant improvements in fibrosis level were found in both the Vein and Artery groups with respect to that in the Control group at 4, 8, and 12 weeks post-BMSC administration. (b) With respect to the change in (Δ) fibrotic area, a significant improvement in fibrosis was found at 4 weeks post-BMSC administration in the Vein (Δfibrotic area: −2.5 ± 1.1%) and Artery groups (−3.8 ± 3.0%) relative to that in the Control group (+0.9 ± 1.0%). A greater effect was found in the Artery group, and this effect was maintained even after 8 (−4.3 ± 3.2%) and 12 (−4.2 ± 2.8%) weeks.
Fig 5.
Therapeutic effects of transfused BMSCs in a CCl4-induced canine liver fibrosis model.
After the induction of liver fibrosis, canines were treated with BMSCs via the peripheral vein (Vein group) or hepatic artery (Artery group). (a) Indocyanine green (ICG) half-life (t1/2) in the Vein group was slightly decreased at 4 weeks post-BMSC administration, from 13.4 ± 2.3 min (0W) to 12.2 ± 2.0 min (4W), but increased from 8 weeks onward to 14.3 ± 1.1 min (8W) and 13.7 ± 1.7 min (12W). In the Artery group, the ICG t1/2 decreased from 16.1 ± 2.3 min (0W) to 13.4 ± 0.8 min (4W), and this value was subsequently maintained (12.3 ± 3.3 min (8W), 12.0 ± 1.5 min (12W)). (b) The ΔICG t1/2 value was significantly decreased in the Vein group compared to that in the Control group at 4 weeks post-BMSC administration (ΔICG t1/2 (min): −1.2 ± 0.7 vs. 1.7 ± 1.4; p < 0.01). Moreover, the ICG t1/2 in the Artery group (n = 4) was significantly decreased at 8 weeks (Δt1/2: −4.2 ± 1.7 min vs. +0.4 ± 2.7 min; p < 0.01) and 12 weeks after BMSC administration (Δt1/2: −4.2 ± 1.7 min vs. +0.4 ± 2.7 min; p < 0.05) even when compared to that in the Vein group. All error bars represent the standard deviation of the mean.
Table 1.
Clinical laboratory test results.
Fig 6.
Expression of liver fibrosis-related genes after the induction of liver fibrosis and BMSC treatment.
After the induction of liver fibrosis, canines were treated with BMSCs via the peripheral vein (Vein group) or hepatic artery (Artery group). The expression of COL1A2, COL3, and TIMP-1 mRNA tended to decrease in the Vein group compared to that the Control group, although the differences were not significant. In the Artery group, the expression levels of COL1A2, COL3, and TIMP-1 were significantly lower than those in the Control group at 4, 8, and 12 weeks. Data show the mean ± standard deviation.
Fig 7.
Contrast-enhanced computed tomography (CE-CT) and photomicrographs of liver tissue samples after BMSC application.
(a) After the infusion of CE-CT, no evidence of liver infarction and the absence of major portosystemic shunts (PSS) were observed. (b) Arterial embolization and biliary necrotic images were not observed in the liver tissues following hematoxylin and eosin staining.
Table 2.
Clinical laboratory test results in the safety evaluation.