Inhibition of hepatic stellate cells by bone marrow-derived mesenchymal stem cells in hepatic fibrosis

Abstract

Background/aims: Therapies involving bone-marrow-derived mesenchymal stem cells (BM-MSCs) have considerable potential in the management of hepatic disease. BM-MSCs have been investigated in regenerative medicine due to their ability to secrete various growth factors and cytokines that regress hepatic fibrosis and enhance hepatocyte functionality. The aim of this study was to determine the antifibrosis effect of BM-MSCs on activated hepatic stellate cells (HSCs) and the mechanism underlying how BM-MSCs modulate the function of activated HSCs.

Methods: We used HSCs in both direct and indirect co-culture systems with BM-MSCs to evaluate the antifibrosis effect of BM-MSCs. The cell viability and apoptosis were evaluated by a direct co-culture system of activated HSCs with BM-MSCs. The activations of both HSCs alone and HSCs with BM-MSCs in the direct co-culture system were observed by immunocytochemistry for alpha-smooth muscle actin (α-SMA). The levels of growth factors and cytokines were evaluated by an indirect co-culture system of activated HSCs with BM-MSCs.

Results: The BM-MSCs in the direct co-culture system significantly decreased the production of α-SMA and the viability of activated HSCs, whereas they induced the apoptosis of activated HSCs. The BM-MSCs in the indirect co-culture system decreased the production of transforming growth factor-β1 and interleukin (IL)-6, whereas they increased the production of hepatocyte growth factor and IL-10. These results confirmed that the juxtacrine and paracrine effects of BM-MSCs can inhibit the proliferative, fibrogenic function of activated HSCs and have the potential to reverse the fibrotic process by inhibiting the production of α-SMA and inducing the apoptosis of HSCs.

Conclusions: These results have demonstrated that BM-MSCs may exert an antifibrosis effect by modulating the function of activated HSCs.

Keywords: Bone-marrow-derived mesenchymal stem cell; Cirrhosis; Hepatic fibrosis.

Figure 1. Immunophenotypes and differentiation potentials of BM-MSCs. (A) The expressions of cell-surface antigens (CD14, CD34, CD45, CD73, and CD105) were evaluated by flow cytometry. (B) BM-MSCs stained positively for endogenous alkaline phosphatase activity, indicating osteogenic differentiation within an osteogenic medium (OM; I), or stained negatively in control medium (CM; II) (×40). BM-MSCs stained positively for lipid droplets, indicating adipogenic differentiation within adipogenic medium (AM; III), or stained negatively in CM (IV) (×200).

Figure 2. Expression of α-SMA in the direct co-culture system of activated HSCs with BM-MSCs as measured by immunocytochemistry. The expression of α-SMA showed that (A, B) activated HSCs and (C, D) activated HSCs with BM-MSCs by fluorescent immunocytochemistry. The expression of α-SMA in cells treated (A, C) with or (B, D) without TGF-β1. Nuclei were stained with DAPI. Merged immunofluorescence images of α-SMA (green) and DAPI (blue). (Scale bar, 75 µm). α-SMA, alpha-smooth muscle actin; HSCs, hepatic stellate cells; BM-MSCs, bone marrow-derived mesenchymal stem cells; TGF-β1, transforming growth factor-beta 1; DAPI, 6-diamino-2-phenylindole.

Figure 3. Cytokine levels of TGF-β1, HGF, IL-6, and IL-10. Indirect co-culture system of activated HSCs with BM-MSCs decreased the production of (A) TGF-β1 and (B) IL-6. Whereas, co-culture system of activated HSCs with BM-MSCs increased the production of (C) HGF and (D) IL-10. Values are presented as mean ± SD. ^*P<0.01. HSCs, hepatic stellate cells; MSCs, mesenchymal stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells.

Figure 4. Inhibition of viability and induction of apoptosis in activated HSCs by BM-MSCs. (A) The HSCs viability was decreased and (B) apoptosis was increased with direct co-culture system of activated HSCs with BM-MSCs. Values are presented as mean ± SD. ^*P<0.01. HSCs, hepatic stellate cells; MSCs, mesenchymal stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells.