Int J Stem Cells.  2019 Nov;12(3):400-409. 10.15283/ijsc18143.

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

• Sabry D
• Mohamed A
• Monir M
• Ibrahim HA

• KMID: 2465880
• DOI: http://doi.org/10.15283/ijsc18143

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 (CCLâ‚„) 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 CCLâ‚„ induced liver fibrosis in rats.

Keyword

Mesenchymal stem cells; Microvesicles; Liver fibrosis

MeSH Terms

Alanine Transaminase
Animals
Bone Marrow
Carbon Tetrachloride
Collagen
Gene Expression
Liver Cirrhosis*
Liver*
Mesenchymal Stromal Cells*
Microscopy, Electron, Transmission
Polymerase Chain Reaction
Rats*
Reverse Transcription
RNA
Serum Albumin
Transforming Growth Factors
Vascular Endothelial Growth Factor A
Alanine Transaminase
Carbon Tetrachloride
Collagen
RNA
Serum Albumin
Transforming Growth Factors
Vascular Endothelial Growth Factor A

Figure

• 

  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.

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  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.

Reference

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