Korean J Hepatobiliary Pancreat Surg.  2013 Feb;17(1):21-33. 10.14701/kjhbps.2013.17.1.21.

Therapeutic potentials occurring during the early differentiation process of mesenchymal stem cells in a rats model with thioacetamide-induced liver fibrosis

• Choi ST
• Hwang S
• Hong HN
• Won YJ
• Ahn CS
• Ha TY
• Song GW
• Jung DH
• Park GC
• Lee SG

• KMID: 2243165
• DOI: http://doi.org/10.14701/kjhbps.2013.17.1.21

Abstract

BACKGROUNDS/AIMS
Mesenchymal stem cells (MSCs) have the capacity to differentiate into hepatocytes, The purpose of this study is to investigate the MSCs' differentiation process and therapeutic potentials by comparing isolated MSCs with HGF-treated MSCs in rat's model with thiacetamide (TAA)-induced cirrhosis.
METHODS
Male Sprague-Dawley (SD) rats, weighing 100-150 g were used in this study. To induce liver fibrosis, recipient rats were taken with 0.04% thioacetamide (TAA) in the drinking water (400 mg TAA/L) for 8 weeks. The rats underlying liver cirrhosis were divided into 3 groups according to the transplanted materials, compared to normal saline as control (I) and isolated MSCs (II) HGF-treated MSCs.
RESULTS
Severe hepatic fibrosis and hepatocyte destruction were detected in the control group. Less hepatic cirrhosis and collagen formation, more hepatocyte regeneration and glycogen storage were detected in isolated MSCs compared to HGF-treated MSCs group, Distribution of red autofluorescence is mainly localized near the sinusoids in isolated MSCs, scattered away the sinusoids in HGF-treated MSCs group. MSCs transdifferentiated into CK-19 postive Oval cells and then to albulmin-producing hepatocytes, HGF treated MSCs differentiated into hepatocyte without the intermediate oval cells phase. HGF treated MSCs became the CK18-positive, MSCs became CD 90-positive.
CONCLUSIONS
Significant hepatocyte differentiation occurred in not HGF-treated MSCs but isolated MSCs group unexpectedly. These results suggest that the beneficial effect of MSCs on in rat's model with TAA-induced cirrhosis may occur during early differentiation course of MSCs. Mature hepatocyte itself has a little effect on the accelerated differentiation and functional capacity of hepatic lineage cell-line.

Keyword

Mesenchymal stem cell; Hepatocyte; Differentiation; Liver cirrhosis; Rat model; Thioacetamide

MeSH Terms

Animals
Collagen
Drinking Water
Fibrosis
Glycogen
Hepatocytes
Humans
Liver
Liver Cirrhosis
Male
Mesenchymal Stromal Cells
Rats
Regeneration
Thioacetamide
Transplants
Collagen
Drinking Water
Glycogen
Thioacetamide

Figure

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  Fig. 1 Histological appearances in the TAA-induced cirrhotic livers. For comparing isolated MSCs group with normal saline group or HGF-treated MSC group, each specimen was stained with H&E stain (Original magnification: ×10, ×20); more prominent hepatocyte regeneration and decreased hepatic fibrosis were detected in isolated MSCs group, but not normal saline or HGF-treated MSCs group.

• 

  Fig. 2 Histological appearances in the TAA-induced cirrhotic livers. For comparing isolated MSCs group with normal saline group or HGF-treated MSC group, each specimen was stained with PAS stain (Original magnification: ×4, ×10, ×20); more prominent hepatocyte regeneration and glycogen storage were detected in isolated MSCs group, but not normal saline or HGF-treated MSCs group.

• 

  Fig. 3 Histological appearances in the TAA-induced cirrhotic livers. For comparing isolated MSCs group with normal saline group or HGF-treated MSC group, each specimen was stained with Sirius-red stain (Original magnification: ×10, ×20); Alleviated septal fibrosis and less collagen deposition were detected in isolated MSCs group, thickened fibrosis and more collagen deposition in normal saline or HGF-treated MSCs group.

• 

  Fig. 4 Western blot analysis and RT-PCR study also showed these serial changes of fibrosis, Quantification of fibrosis induction and MSC-associated effects were assessed through antibody studies for collagen α-1 and fibronectin and RT-PCR for collagnen α-1 messanger ribonucleic andi (mRNA). The expressed amount of collagen α-1 and fibronectin were increased in 8 weeks after TAA ingestion and more decreased in additional 2 weeks after MSC infusion, contrary to control group.

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  Fig. 5 Fluorescence microscope image showed that stem cells differentiate to hepatocyte and express the albumin in 2 weeks after HGF treatment. (A) Stem cells themselves without differentiation to hepatocyte-like cell (CELL STALKER-CSR dye staining, red; Original magnification: ×10, ×20). (B) Stems cells with differentiation to hepatocyte and albumin expression (Anti-albumin staining, green; Original magnification: ×10, ×20).

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  Fig. 6 Western blot analysis indicated that these specific markers were actively synthesized before and after hepatic differentiation: HGF-treated MSCs became the CK18-positive, Isolated MSCs became CD 90-postive. CD90 is a stem cell marker and CK18 is a hepatocyte marker.

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  Fig. 7 Transplanted donor cell distribution to the recipient livers. High fluorescence microscope images (×20). (A) A number of Red autofluorescence is mainly localized near hepatic sinusoids in stem cell group, which indicated better engraftment (B). A few red autofluorescence is localized near peripheral hepatic sinusoids in HGF-treated stem cells group, indicated poor engraftment.

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  Fig. 8 Transplanted donor cell engraftment to the recipient liver and differentiation into the hepatocytes. High fluorescence microscope images (×20). Anti-CK19 staining, green. MSCs with CELL STALKER-CSR dye staining, red. Albumin positive stem cells in the merged images, yellow. Hepatogenic differentiation-related analyses showed that infusion of isolated MSCs advanced the appearance of oval cells in 1 week, leading to a higher population of hepatic progenitor cells and a lower population of MSCs at 3 weeks.

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  Fig. 9 Transplanted donor cell engraftment to the recipient liver and differentiation into the hepatocytes. High fluorescence microscope images (×20). Hepatogenic differentiantion of implanted MSCs was assessed by immunohistochemistry using Antibody to albunin, CK-19 and CELL STALKKER-CSR dye to assess the MSCs. One week after MSC implantation, CK19 stained oval cells derived from MSCs were located around the portal veins. At 3 weeks, more differentiated MSCs or hepatocyte-like cells were located toward the hepatic parenchyma, and albumin-stained areas had expanded and overlapped the MSC-stained areas. Nearly all the CK19/thy1- and albumin-stained areas overlapped the MSC-stained areas, indicating that most MSCs had transdifferentiated into oval progenitor cells and then into functional hepatocyte-like cells after hepatic implantation.

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  Fig. 10 Hepatic fibrosis and repair by transplanted donor cell engraftment to the recipient liver. High fluorescence microscope images: Engrafted isolated MSC groups express lower α-SMA and higher TUNEL compared with HGF-treated MSCs group. (A) Anti-α-SMA staining, red. (B) TUNEL staining, green. (C) α-SMA and TUNEL positive stem cells in the merged images, yellow. The merged images of TUNEL and α-SMA stains also revealed that apoptosis of hepatic stellate cells was less evident after HGF treatment.

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