The effectiveness of CXCL9-expressing UC-MSCs in suppressing liver fibrosis and stimulating liver regeneration in vitro and in vivo shows that CXCL9-UC-MSCs have a powerful therapeutic effect in rats with CCl4-induced liver fibrosis. The major findings are as follows:(1) The expression of CXCL9 did not affect the characterization of UC-MSCs, nor does it affect the ability of UC-MSCs to differentiate into hepatocyte like cells, but had an effect on the growth and proliferation of UC-MSCs; (2) transplantation of CXCL9-UC-MSCs greatly ameliorated liver injury, and the effects of CXCL9-UC-MSCs exceeded those of control UC-MSCs; (3) treatment with CXCL9-UC-MSCs reversed liver damage and decreased toxicity, seen as reduction of ALT, AST and T-Bil back to normal levels; and (4) CXCL9-UC-MSCs homed into fibrosis-affected rat liver more efficiently than control UC-MSCs.
Transplantation of liver progenitor cells (LPCs), mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) has become a promising therapy to treat various liver disease mainly due to the ability of these cell types to promote liver regeneration [9,10]. Among the that, MSCs have the characteristics of multi-directional differentiation potential, low immunogenicity, and non-induction of tumorigenesis even after multiple passages in vitro [11-13]. Human MSCs, can be derived from bone marrow (BM-MSCs), adipose tissue (AT-MSCs), amniotic fluid (AF-MSCs), dental pulp (DP-MSCs), umbilical cord (UC-MSCs) and fetal lung (FL-MSCs) [14]. UC-MSCs and BM-MSCs are widely used in liver injured diseases, but UC-MSCs have the advantages of convenient source, lower immunogenicity and less ethical restrictions compare to BM-MSCs. Therefore, UC-MSCs are the cell source in this study.
Most chronic liver diseases involving inflammation have a fibrotic component which involves remodeling and excess accumulation of extracellular matrix components (EMC), and fibrosis leads to liver failure and death eventually. In this study, the liver fibrosis rat model was generated by intraperitoneal injection of CCL4 as previously reported [15]. In rencent years, MSCs transplantation has been considered as a promising approach for the treatment of liver fibrosis [16-18]. In vitro and in vivo experiments demonstrated that MSCs mainly exert antifibrotic effects by paracrine mechanisms [16]. MSCs can produce a range of cytokines and growth factors, such as insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and milk fat globule-EGF factor-8 (MFGE8) which have been shown to promote hepatocyte regeneration in situ, inhibit apoptosis of hepatocyte, suppress inflammatory responses, affect HSC activation, and promote the degradation of extracellular matrix [19-22]. Moreover, MSCs can differentiate into hepatocyte-like cells and are suitable for transfection with exogenous genes [23]. However, the treatment effect of MSCs transplantation is not very satisfactory, the main reason is lacking of enough cells to function in the injured sites of liver after injected into the body.
The CXCL9 is a famous member of the CXC chemokine family and a potent chemoattractant for activated T lymphocytes, natural killer cells and monocytes, participating recruitment of Th1 regulatory T cells[24]. In addition to immune responses of Th1, CXCL9 and its receptor, CXCR3 has also been reported to be involved in the occurrence of chronic diseases in lung and liver [7,25,26-29]. As we known, the occurrence of liver fibrosis is closely related to the activation and proliferation of HSC and the up-regulation of the expression of vascular endothelial growth factor (VEGF). Activated HSCs are the major source of collagen in the liver, which can secrete a large amount of ECM protein, tissue inhibitors of metalloproteinases and matrix metalloproteinases (MMPs), causing the remodeling of liver structure subsequently[30,31]. Interestingly, increasing concentrations of CXCL9 lead to dose-dependent down-regulation of transforming growth factor β1 (TGF-β1)and collagen 1α1mRNA, which are playing an important role in the process of liver forosis. Furthermore, collagen protein expression of stellate cells can be strongly suppressed by CXCL9 [7]. VEGF is a central angiogenic factor during chronic liver injury. In the process of fibrosis formation, VEGF promotes the formation of new blood vessels and accelerates the remodeling process of liver lobules[32]. However, CXCL9 can be used as an anti-fibrotic cytokine together with metalloproteinase 13 (MMP13) to achieve an anti-fibrotic effect by regulating the expression of VEGF[33]. Recently, some studies have shown that the CXCL9/CXCR3 signaling pathway can promote cell migration, and proposed that MSCs could interact with aortic endothelial cells showing firm adhesion, and crawling and spreading behaviour, which were all enhanced by CXCL9 [34,35]. But some researchers suggested that the antifibrotic action of CXCL9 might not be strong enough to ultimately cause a reduction in fibrosis in human diseases[7]. Therefore, we hypothesized that overexpression of CXCL9 could improve the therapeutic efficacy of UC-MSCs transplantation for the treatment of liver fibrosis, and consequently promote the homing capabilities of UC-MSCs to the rat livers affected by fibrosis.
The purpose of this study is to combine UC-MSCs and chemokines with genetic engineering technology, so that they can play a stronger role in anti-fibrosis. Extensive studies have suggested that gene therapy is likely to become an option for the treatment of many forms of liver diseases, and may ameliorate patient care, survival, and prognosis[36,37]. Lentiviral vector is one of the most widely used gene delivery system, the vector can effectively deliver foreign genes into host chromosomes, so as to achieve stable expression. In terms of infection ability, it can highly efficient infect neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells and other types of cells, so as to achieve good gene therapy effect[38]. Considering the advantages of the lentivirus as a highly efficient gene transfection system for gene therapy, the lentivirus vector was selected in this study to deliver the CXCL9 gene into UC-MSCs. Our results validated that after puromycin screening, the transfected UC-MSCs could persistently expressing CXCL9.
In this study, the cell therapy and gene therapy were combined to achieve a better anti-fibrosis effect. And this combination will not affect the characteristics of UC-MSCs, such as the ability to differentiate into hepatocytes. As expected, when the CXCL9-UC-MSCs and control UC-MSCs transplantation groups were compared, we found that liver fibrosis rats in the CXCL9-UC-MSCs transplantation group experienced better therapeutic effects as assessed by markers for liver function and liver pathology. The liver function of the CXCL9-UC-MSCs group was significantly improved, and the fibrosis was also obviously different than that of the control UC-MSCs group. Besides, we detected the levels of liver enzymes such as ALT, AST and T-Bil in serum 4 weeks after the initiation of cell therapy, and found that their levels were notablely decreased in both the CXCL9-UC-MSCs and control UC-MSCs groups. The AST, AST and T-Bil levels decreased to normal levels in the CXCL9-UC-MSCs-treated group, whereas they were slightly higher than normal in the control UC-MSCs-treated group. Moreover, from the results of frozen sections, the homing ability of CXCL9-UC-MSCs and the residence time in the liver are indeed stronger and longer than those of the control UC-MSCs group. These results clearly indicated that the transplantation of UC-MSCs have a therapeutic effect, and the transplantation of CXCL9-UC-MSCs has a synergistic regenerative effect on fibrotic livers. Regeneration may be related to the promotion of UC-MSCs migration and incorporation into liver tissue.
This study still has several limitations. First, CXCL9 has undermined the growth and proliferation of UC-MSCs, further studies are underway in our laboratory to characterize CXCL9-UC-MSCs-secreted cytokines in cell culture. Second, we have only observed that the CXCL9-expressing UC-MSCs can improve liver fibrosis better, but we have not discussed the exact mechanism yet. At last, the microenvironment of liver fibrosis cannot be fully replicated in vitro, so whether CXCL9 affects the differentiation of UC-MSCs into hepatocyte like cells in vivo needs to be further explored.