Effects of human amnion-derived mesenchymal stem cells and conditioned medium in rats with sclerosing cholangitis

Abstract

Mesenchymal stem cells (MSCs) represent a valuable cell source in regenerative medicine, and large numbers of MSCs can be isolated from the amnion noninvasively. Sclerosing cholangitis is a chronic cholestatic disease and characterized by progressive biliary destruction leading to cirrhosis. Many factors are involved in the development of sclerosing cholangitis; however, effective medical therapy is not established. We investigated the effects of human amnion-derived MSCs (hAMSCs) and conditioned medium (CM) obtained from hAMSC cultures in rats with sclerosing cholangitis. Sclerosing cholangitis was induced via the intragastric administration of 100 mg/kg alpha-naphthylisothiocyanate (ANIT) twice weekly for 4 weeks. One million hAMSCs or 200 μL of CM were intravenously administered on days 15 and 22. Rats were sacrificed on day 29 and evaluated via histological, immunohistochemical, and mRNA expression analyses. hAMSC transplantation and CM administration significantly improved the histological score. In addition, these two interventions significantly improved biliary hyperplasia, peribiliary fibrosis, and inflammation in Glisson's sheath. Accordingly, CK19, MMP-9, and TNF-α, and MCP-1 expression in the liver was also decreased by hAMSC and CM administration. In conclusion, hAMSC and CM administration ameliorated biliary hyperplasia, peribiliary fibrosis, and inflammation in a rat model of sclerosing cholangitis. hAMSCs and CM may represent new modalities for treating sclerosing cholangitis.

Keywords: Mesenchymal stem cells; alpha-naphthylisothiocyanate; amnion; regenerative medicine; sclerosing cholangitis.

Figure 1

Experimental protocol for creating an alpha-naphthylisothiocyanate (ANIT)-induced sclerosing cholangitis model. Rats received 100 mg/kg ANIT twice weekly for 4 weeks. One million human amnion-derived mesenchymal stem cells (hAMSCs) or 200 μL of conditioned medium (CM) were intravenously administered on days 15 and 22. All rats were sacrificed on day 29.

Figure 2

Effect of human amnion-derived mesenchymal stem cells (hAMSCs) and conditioned medium (CM) obtained from hAMSCs on histological parameters. A. Hematoxylin and eosin staining. Glisson’s sheath (upper) and necrotic lesions (lower, arrows) are shown. Scale bars, 100 μm. B. Liver histology scores regarding biliary hyperplasia, fibroblast proliferation and neutrophilic infiltration in Glisson’s sheath in 10 sections per sample in each high-power field. C. Number of necrosis lesions in whole field (WF). Values are expressed as the mean ± SEM (N = 6-10 animals/group). **P < 0.01 vs. the control group. †P < 0.05 and ††P < 0.01 vs. the alpha-naphthylisothiocyanate (ANIT) group.

Figure 3

Effects of human amnion-derived mesenchymal stem cells (hAMSCs) and conditioned medium (CM) obtained from hAMSCs on biliary hyperplasia. A. Cytokeratin 19 (CK19) expression. B. Quantitative reverse transcription-polymerase chain reaction for CK19. The stained areas were measured from 10 sections per sample in each low-power field. Scale bars, 100 μm. Values are expressed as the mean ± SEM (N = 6-10 animals/group). *P < 0.05 and **P < 0.01 vs. the control group. †P < 0.05 vs. the alpha-naphthylisothiocyanate (ANIT) group.

Figure 4

Effects of human amnion-derived mesenchymal stem cell (hAMSCs) and conditioned medium (CM) obtained from hAMSCs on fibrosis, collagen deposition, and fibrosis-related marker expression. A. Sirius Red staining. B. α-smooth muscle actin (SMA) expression. C. Type I collagen expression. D. Quantitative reverse transcription-polymerase chain reaction. Stained areas were measured from 10 sections per sample in each low-power field. Scale bars, 100 μm. Values are expressed as the mean ± SEM (N = 6-10 animals/group). *P < 0.05 and **P < 0.01 vs. the control group. †P < 0.05 and ††P < 0.01 vs. the alpha-naphthylisothiocyanate (ANIT) group.

Figure 5

Effects of human amnion-derived mesenchymal stem cell (hAMSCs) and conditioned medium (CM) obtained from hAMSCs on the infiltration of inflammatory cells and inflammatory mediators. A. CD68 expression. B. Quantitative reverse transcription-polymerase chain reaction. The number of positive cells was counted in 10 sections per sample in each high-power field. Scale bars, 20 μm. Values are expressed as the mean ± SEM (N = 6-10 animals/group). *P < 0.05 and **P < 0.01 vs. the control group. †P < 0.05 and ††P < 0.01 vs. the alpha-naphthylisothiocyanate (ANIT) group.