Wharton's jelly-derived mesenchymal stem cells combined with praziquantel as a potential therapy for Schistosoma mansoni-induced liver fibrosis

Abstract

Liver fibrosis is one of the most serious consequences of S. mansoni infection. The aim of the present study was to investigate the potential anti-fibrotic effect of human Wharton's jelly-derived mesenchymal stem cells (WJMSCs) combined with praziquantel (PZQ) in S. mansoni-infected mice. S. mansoni-infected mice received early (8(th) week post infection) and late (16(th) week post infection) treatment with WJMSCs, alone and combined with oral PZQ. At the 10(th) month post infection, livers were collected for subsequent flow cytometric, histopathological, morphometric, immunohistochemical, gene expression, and gelatin zymographic studies. After transplantation, WJMSCs differentiated into functioning liver-like cells as evidenced by their ability to express human hepatocyte-specific markers. Regression of S. mansoni-induced liver fibrosis was also observed in transplanted groups, as evidenced by histopathological, morphometric, and gelatin zymographic results besides decreased expression of three essential contributors to liver fibrosis in this particular model; alpha smooth muscle actin, collagen-I, and interleukin-13. PZQ additionally enhanced the beneficial effects observed in WJMSCs-treated groups. Our results suggest that combining WJMSCs to PZQ caused better enhancement in S. mansoni-induced liver fibrosis, compared to using each alone.

Figure 1. Immunophenotype of WJ-derived cells.

Cells were labeled with FITC- or PE-conjugated antibodies. Shaded areas show the expression of (A) CD29, (B) CD44, (C) CD59, (D) CD73, (E) CD90, (F) CD105, (G) CD14, (H) CD19, (I) CD34, (J) CD45, and (K) HLA-DR.

Figure 2

Photomicrographs of (A) haematoxylin and eosin (H&E, X100) and (B) Sirius red staining (SR, X100), as well as immunohistochemical staining (immunostain, DAB, X400) of (C) cytokeratin-7 (CK-7) and (D) oval cell marker type 6 (OV-6) of liver sections of control and treated groups. In the H&E- and SR-stained sections, fibrosis was less notable in the groups which received WJMSCs combined with PZQ where a reduction in granuloma diameters was observed. The fibrotic areas stained with Sirius red was significantly less in the groups which received combined treatment. As for the immunostained sections, no expression was observed in infected control and PZQ-treated groups. However, positive expression (brown cytoplasmic discolouration) was observed in all transplanted groups.

Figure 3

Photomicrographs of immunohistochemical staining (immunostain, DAB, X400) of (A) human alpha fetoprotein (AFP), (B) cytokeratin-18 (CK-18), (C) Hep par-1, (D) alpha smooth muscle actin (α-SMA), and (E) vimentin in liver sections of control and treated groups. No expression was observed in infected control and praziquantel (PZQ)-treated groups, whereas newly-formed liver-like cells in groups treated with Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) showed positive expression (brown cytoplasmic discolouration) for AFP, CK-18, and Hep par-1. Positive expression of α-SMA and vimentin was observed only in scattered liver-like cells in groups treated with WJMSCs with the least expression observed in the groups which received WJMSCs combined with PZQ.

Figure 4

Effects of treatment with Wharton’s jelly-derived mesenchymal stem cells (WJMSCs), given either alone or combined with praziquantel (PZQ), on the immunohistochemical expression of human (A) alpha fetoprotein (AFP), (B) cytokeratin-7 (CK-7), (C) CK-18, (D) Hep par-1, (E) oval cell marker type-6 (OV-6), (F) alpha smooth muscle actin (α-SMA), and (G) vimentin in the liver sections of mice infected with S. mansoni. WJMSCs (1.5 × 10⁶ cells/mouse) were injected at either the 8^th (early) or 16^th (late) week (W) post infection. PZQ (500 mg/kg/day) was orally given at the 7^th W post infection for 2 consecutive days. Animals were sacrificed at the 10^th month post infection. Values are presented as means ± S.E. (n = 8–10). Significantly different (P < 0.05) ^£ versus WJMSCs (8W), ^@ versus WJMSCs (16W), and ^† versus PZQ + WJMSCs (8W). Statistical analysis was performed by one-way analysis of variance (One-way ANOVA) followed by Bonferroni post hoc test. ^O Significant interaction when PZQ and WJMSCs were combined using factorial design.

Figure 5

Effects of treatment with Wharton’s jelly-derived mesenchymal stem cells (WJMSCs), given either alone or combined with praziquantel (PZQ), on the gene expression of human (A) albumin (Alb) and (B) alpha fetoprotein (AFP) and murine (C) alpha smooth muscle actin (α-SMA), (D) collagen-I (Col-I), and (E) interleukin-13 (IL-13) in the liver tissue of mice infected with S. mansoni. WJMSCs (1.5 × 10⁶ cells/mouse) were injected at either the 8^th (early) or 16^th (late) week (W) post infection. PZQ (500 mg/kg/day) was orally given at the 7^th W post infection for 2 consecutive days. Animals were sacrificed at the 10^th month post infection. Values are presented as means ± S.E. (n = 8-10). Significantly different (P < 0.05) ^* vs infected control, ^$ vs PZQ, ^£ vs WJMSCs (8W), ^@ vs WJMSCs (16W), and ^† vs PZQ + WJMSCs (8W). Statistical analysis was performed by one-way analysis of variance (One-way ANOVA) followed by Bonferroni post hoc test. ^O Significant interaction when PZQ and WJMSCs were combined using factorial design.

Figure 6. Effects of treatment with Wharton’s jelly-derived mesenchymal stem cells (WJMSCs), given either alone or combined with praziquantel (PZQ), on gelatin zymography of matrix metalloproteinase (MMP)-2 and -9 in the liver tissue of mice infected with S. mansoni.

Quantitative analysis of the average relative band areas of (A) MMP-2 and (B) MMP-9. (C) Representative gelatin zymography showing the effects of different treatments on MMP-2 and -9 enzyme activities. WJMSCs (1.5x10⁶ cells/mouse) were injected at either the 8^th (early) or 16^th (late) week (W) post infection. PZQ (500 mg/kg/day) was orally given at the 7^th W post infection for 2 consecutive days. Animals were sacrificed at the 10^th month post infection. Values are presented as means ± S.E. (n = 8–10). Significantly different (P < 0.05) ^* vs infected control, and ^$ vs PZQ. Statistical analysis was performed by one-way analysis of variance (One-way ANOVA) followed by Bonferroni post hoc test.