The Effect of Mesenchymal Stem Cells Derived Microvesicles on the Treatment of Experimental CCL4 Induced Liver Fibrosis in Rats

Abstract

Background and objectives: The release of microvesicles (MVs) from mesenchymal stem cells (MSCs) has been implicated in intercellular communication, and may contribute to beneficial paracrine effects of stem cell-based therapies. We investigated the effect of administration of MSC-MVs on the therapeutic potential of carbon tetrachloride (CCL₄) induced liver fibrosis in rats.

Methods: Our work included: isolation and further identification of bone marrow MSC-MVs by transmission electron microscopy (TEM). Liver fibrosis was induced in rats by CCl4 followed by injection of prepared MSC-MVs in injured rats. The effects of MSC-MVs were evaluated by biochemical analysis of liver functions, RNA gene expression quantitation for collagen-1α, transforming growth factor β (TGF-β), interleukin-1β (IL-1β), vascular endothelial growth factor (VEGF) by real time reverse transcription PCR (RT-PCR) techniques. Finally histopathological examination of the liver tissues was assessed for all studied groups.

Results: BM-MSC-MVs treated group showed significant increase in serum albumin levels, VEGF quantitative gene expression (p<0.05), while it showed a significant decrease in serum alanine transaminase (ALT) enzyme levels, quantitative gene expression of TGF-β, collagen-1α, IL-1β compared to CCL₄ fibrotic group (p<0.05). Additionally, the histopathological assessment of the liver tissues of BM-MSC-MVs treated group showed marked decrease in the collagen deposition & improvement of histopathological picture in comparison with CCL₄ fibrotic group.

Conclusions: Our study demonstrates that BM-MSC-MVs possess anti-fibrotic, anti-inflammatory, and pro-angiogenic properties which can promote the resolution of CCL₄ induced liver fibrosis in rats.

Keywords: Liver fibrosis; Mesenchymal stem cells; Microvesicles.

Fig. 1

BM-MSCs in culture. (A) After 24 hour with black arrow to identify MSCs as fibroblast like cell (100×), (B) after 72 hour (100×), (C) blue arrow to show MSCs during their cell division (400×) and (D) after 14 days to show 80% confluence proliferating MSCs. FACS analysis for cultured MSCs. They were negative for CD34⁻ (0.2%), CD45⁻ (0.07%) and positive for CD29⁺ (99.72%), CD105⁺ (98.4%), CD73⁺ (95.7%) and CD90⁺(98.5%). (E) MSCs were differentiated into osteoblasts stained with Alizarin Red S stain (black arrow) (100×). (F) MSCs were differentiated into chondrocytes stained with Alcian blue stain (black arrow) (100×).

Fig. 2

(A) Western blots scanning densitometry for CD61, CD80 and CD83 for 3 different passages of MVs derived BM-MSCs. (B) TEM of MVs; they were spheroid (━ 500 nm) and have well defined membrane with heterogeneous contents.

Fig. 3

(A) Serum Albumin levels (g/dl) in all studied groups. (B) Serum ALT levels (U/l) in all studied groups.

Fig. 4

(A) qRT-PCR genes expression of collagen-1α, (B) TGF-β, (C) VEGF and (D) IL-1β in liver tissues of all studied groups.

Fig. 5

Divided into 3 panels. First panel: Histopathology of liver tissue in control group stained with H&E: a (40×), b (100×) and c (400×) showing normal hepatic architecture, normal hepatocytes, portal tracts and central veins. Second panel: (A) Histopathology of liver tissue in CCL₄ fibrotic group stained with H&E: a (40×) and b (100×) showing loss of normal hepatic architecture, marked fibrous septations with complete nodules formation and fibrous expansion of the portal tracts, c (400×) showing dysplastic changes of hepatocytes in the form of nuclear hyperchromasia and pleomorphism (green arrows), d (400×) showing hydropic degeneration of hepatocytes (red arrows) and periportal lymphocytic infiltration (orange arrows). (B) Histopathology of liver tissue in CCL₄ fibrotic group stained with Sirius red: a (40×), b (100×) showing marked fibrous septations with complete nodules formation, c (400×) showing fibrous expansion of the portal tract. Third panel: Histopathology of liver tissue in BM-MSC-MVs treated group stained with H&E: a (40×), b (100×) showing preserved liver architecture and no fibrosis, c (400×) showing thin plate of normal hepatocytes and no inflammation. Sirius red staining: d (100×) showing no fibrosis & the Sirius red stain is only limited to the outline of blood vessels.