Int J Stem Cells.  2021 Nov;14(4):475-484. 10.15283/ijsc21031.

Human Liver Stem Cell Transplantation Alleviates Liver Fibrosis in a Rat Model of CCl ₄ -Induced Liver Fibrosis

• Lee JH
• Lee S
• Park HJ
• Kim YA
• Lee SK

• KMID: 2522565
• DOI: http://doi.org/10.15283/ijsc21031

Abstract

Background and Objectives
Mesenchymal stem cells (MSCs) elicit therapeutic effects against liver fibrosis in animal models. Human liver stem cells (HLSCs) are cells isolated from human liver tissue that have mesenchymal morphology and express MSC markers. HLSCs also possess intrahepatic stem cell properties. We introduce a rat model of liver fibrosis and trans-portal transplantation of HLSC to demonstrate alleviation of liver fibrosis.
Methods and Results
Liver fibrosis was induced by intraperitoneal injection of Carbon tetrachloride (CCl ₄ ). Sprague Dawley rats underwent simultaneous partial hepatectomy of the left hepatic lobe and HLSC transplantation via the portal vein. Gross appearance of the liver observed following CCl ₄ injection showed cholestasis and surface nodularity. Sirius red staining revealed deposition of collagen fibers in the extracellular matrix (ECM). Following HLSC transplantation, human albumin secreting cells were detected by immunohistochemistry in liver specimens. Quantitative measurements of fibrosis area stained by Sirius red were compared between baseline and post-HLSC transplant (1×10 ⁷ cells) following 10 weeks of CCl ₄treatment liver specimens. Fibrosis area (p＜0.05), serum markers of liver inflammation and fibrosis (AST, ALT levels and APRI, p＜0.05) significantly decreased from baseline after HLSC transplantation. RNA expression in liver tissues revealed significant decrease in tissue inhibitor of matrix metalloproteinase 1 (TIMP1), TIMP2 expression and increase in hepatocyte growth factor expression following HLSC transplantation (p＜0.05).
Conclusions
HLSC transplantation effectively reduced the area of liver fibrosis with increased expression of factors promoting ECM degradation. These findings suggest the potential therapeutic role of HLSCs in various liver diseases presenting with liver fibrosis.

Keyword

Human liver stem cell; Human liver stem cell; Liver fibrosis; Liver fibrosis; Stem cell transplantation; Stem cell transplantation; Mesenchymal stem cell; Mesenchymal stem cell

Figure

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  Fig. 1 Scheme for the generation of rat liver fibrosis model and HLSC transplantation. SD rats received biweekly intraperitoneal injections of CCl4 0.65∼1.0 ml/kg for up to 10 weeks. HLSCs were isolated from human liver grafts unsuitable for transplantation, as described in ‘Methods’. Rats underwent abdominal surgery trans-portal HLSC transplantation with simultaneous left hepatic lobectomy.

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  Fig. 2 (A) Therapeutic effects of HLSC transplantation in CCl4-induced liver fibrosis in rat model. Liver tissues were obtained from rats at 10 weeks (baseline) and at 14 weeks after initiation of CCl4 treatment (post 14w CCl4). Extracellular deposition of collagen fibers was stained with Sirius red. The presence of the transplanted HLSCs in the liver tissue was identified by immunohistoche-mistry using antibodies against human albumin (hALB). (B) Real-time qPCR analyses of ECM remodeling factors in rat liver specimens following two fibrosis induction protocols. Significant increase in MMP2, HGF expression and decrease in TIMP1, TIMP2 expression are seen following HLSC transplantation with intense toxicity protocol. These changes are not consistently observed in control groups.

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  Fig. 3 (A) Changes in fibrosis area following two fibrosis induction protocols. Significant decrease in fibrosis area compared to baseline is seen at 14 weeks after liver fibrosis induction when HLSC transplantation is performed. In the control group, fibrosis area is increased from baseline to week 14 when CCl4 is given with the intense toxicity protocol. In the moderated toxicity protocol, spontaneous regression of liver fibrosis is observed at 14 weeks. (B) Changes in fibrosis area according to transplantation of three HLSC strains. Significant decrease in fibrosis area compared to baseline is seen at 14 weeks after liver fibrosis induction with HLSC7 transplantation. No significant change is seen in the other two HLSC strains. In the control group, fibrosis area is increased from week 10 to week 14. (C) Changes in fibrosis area following three transplantation schemes. Significant decrease in fibrosis area compared to baseline is seen in HLSCx (standard transplantation scheme) 14 weeks after liver fibrosis induction. No significant change is seen in the other two transplant schemes. In the control group, fibrosis area is increased from week 10 to week 14.

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  Fig. 4 (A) Therapeutic effects of HLSC transplantation with various HLSC strains. Each strain of HLSCs were transplanted following 10 weeks of CCl4 treatment. Liver tissues were obtained from rats at 10 weeks (baseline) and at 14 weeks after initiation of CCl4 treatment (post 14w CCl4). Extracellular deposition of collagen fibers was stained with Sirius red. Histopathological changes of liver sections were observed by staining with hematoxylin and eosin (H&E). The presence of the transplanted HLSCs in the liver tissue was identified by immunohistoche-mistry using antibodies against human albumin (hALB). (B) Real-time qPCR analyses of ECM remodeling factors in rat liver specimens according to transplantation of three HLSC strains. Significant increase in MMP2, HGF expression and decrease in TIMP1, TIMP2 expression is seen following transplantation with HLSC7. These changes are not consistently observed in HLSC15 or HLSC26 stains.

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  Fig. 5 (A) Therapeutic effects of HLSC transplantation according to transplant schemes. HLSCx group received a single transplant of 1×107 cells. HLSC2x group received a single transplant of 2×107 cells. HLSCxx group received 2 HLSC transplants with 1×107 cells, 2 weeks apart. Liver tissues were obtained from rats at 10 weeks (baseline) and at 14 weeks after initiation of CCl4 treatment (post 14w CCl4). Extracellular deposition of collagen fibers was stained with Sirius red. Histopathological changes of liver sections were observed by staining with hematoxylin and eosin (H&E). The presence of the transplanted HLSCs in the liver tissue was identified by immunohistoche-mistry using antibodies against human albumin (hALB). (B) Real-time qPCR analyses of ECM remodeling factors in rat liver specimens following three transplantation schemes. Significant increase in MMP2 and HGF expression is seen following HLSC transplantation. Significant decrease in TIMP1 and TIMP2 expression is seen following HLSC transplantation. These changes are not consistently observed in HLSC2x or HLSCxx schemes.

Reference

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