Effectiveness of human adipose tissue-derived mesenchymal stem cells expressing alpha-1 antitrypsin gene in liver fibrosis: a study in mice

Abstract

Aim: The present study examined the protective potential of human adipose tissue-derived mesenchymal stem cells (hASCs) modified to overexpress alpha-1 antitrypsin (AAT), in a mouse model of the liver fibrosis.

Background: For the treatment of end-stage liver diseases, cell therapy has emerged as a promising noninvasive alternative to liver transplantation. Mesenchymal stem cells (MSCs) are being evaluated due to their dual capabilities of promoting liver regeneration and modulating the pathogenic inflammation of the immune system.

Methods: Liver fibrosis was induced in mice via the intraperitoneal injection of carbon tetrachloride (CCl4). MSCs were extracted from the human adipose tissue. After stemness confirmation, the cells were transduced with the lentiviruses containing the AAT gene, and then injected into the mice's tail vein. Fourteen days' post-transplantation, mice were sacrificed, and blood and tissue samples were collected for analysis. Important liver enzymes, including alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, and total bilirubin (TB), were measured. Histological studies were carried out using the hematoxylin and eosin (H&E), as well as Masson's trichrome (MT) staining.

Results: Compared to hASCs, treatment with AAT-hASCs resulted in greater reductions in ALT, AST, ALP, and TB, as well as normalized albumin levels. AAT-hASCs promoted enhanced liver regeneration histologically, likely attributable to anti-inflammatory and anti-proteolytic properties of AAT.

Conclusion: These findings indicate AAT-engineered hASCs as a promising cell-gene therapy candidate for further study in liver cirrhosis models.

Keywords: Adipose tissue-derived mesenchymal stem cells; Alpha-1 antitrypsin; Carbon tetrachloride; Lentiviral vectors; Liver fibrosis.

Figure 1

A) Adipocyte and osteocyte differentiation of isolated hASCs. (1) The intracellular accumulated lipid droplets show adipogenic differentiation, (2) the intracellular calcium deposits show osteogenic differentiation, and (3) the undifferentiated hASCs serve as a negative control. B) Flow cytometry of hASCs surface marker was positive for CD90 (95.5%), CD105 (96.9%), and CD44 (97.1%) but negative for CD45 (0.769%) and CD34 (0.838%).

Figure 2

Transduced hASCs stably expressing AAT and Jred. (A) The transduction efficacy of transduced hASCs was assessed using a fluorescent microscope and based on the expression of Jred. (B) qPCR expression analysis of AAT expression levels indicated 80-fold increase in the AAT-hASCs. The expression levels were normalized to β-actin, and the values are expressed as mean±SEM (n=3).

Figure 3

Histopathological analysis of livfer fibrosis. (A) H&E staining, and (B) MT staining of the liver tissue slides. (C) Using ImageJ software, a quantitative analysis of liver fibrosis in MT-stained sections was performed. Treatment of liver fibrosis with hASCs and AAT-hASCs had significant positive effects (p<0.05) on the reduction of fibrosis. The values are presented as mean±SEM (n=3).

Figure 4

Serological Liver function markers. ALT, ALP, AST, albumin, and TB concentrations were measured in experimental groups. One-way ANOVA was applied to determine the significance of values in the groups (n=3 per group).