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

Jang YO, Jun BG, Baik SK, Kim MY, Kwon SO - Clin Mol Hepatol (2015)

Bottom Line: Therapies involving bone-marrow-derived mesenchymal stem cells (BM-MSCs) have considerable potential in the management of hepatic disease.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.These results have demonstrated that BM-MSCs may exert an antifibrosis effect by modulating the function of activated HSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea. ; Cell Therapy and Tissue Engineering Center, Yonsei University Wonju College of Medicine, Wonju, Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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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).

Mentions: The immunophenotypes of the BM-MSCs (CD14, CD34, CD45, CD73, and CD105) were analyzed prior to experimental use, and their differentiation potentials were determined (osteogenesis and adipogenesis; Fig. 1). For the immunophenotype analysis, BM-MSCs were stained with antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): CD14-FITC, CD34-FITC, CD45-FITC, CD73-PE, and CD105-PE (BD Biosciences, San Jose, CA, USA). Briefly, 5×105 cells were resuspended in 0.2 mL of phosphate-buffered saline (PBS) and incubated with FITC- or PE-conjugated antibodies for 20 min at room temperature. FITC- or PE-conjugated mouse IgGs were used as the control isotype at the same concentration as the specific primary antibodies. The fluorescence intensity of the cells was evaluated through flow cytometry (Epics XL; Beckman Coulter, Miami, FL, USA). Osteogenic differentiation was determined by first plating the cells at 2×104 cells/cm2 in six-well plates and then incubating them in the following osteogenic medium for 2-3 weeks: DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS, 10 mM β-glycerophosphate, 10-7 M dexamethasone, and 0.2 mM ascorbic acid (Sigma-Aldrich).20 The osteogenic differentiation was quantified from the release of p-nitrophenol from p-nitrophenyl phosphate by the enzyme alkaline phosphatase.21 For adipogenic differentiation, BM-MSCs were plated at 2×104 cells/cm2 in six-well plates and cultured for 1 week; then, differentiation was induced with adipogenic medium (10% FBS, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 µg/mL insulin, and 100 µM indomethacin in high-glucose DMEM) for an additional 3 weeks. The differentiated cells were fixed in 4% paraformaldehyde for 10 min and stained with fresh Oil Red-O solution (Sigma-Aldrich) to display lipid droplets (Fig. 1B). The criteria regarding the use of MSCs included viability greater than 80%; the absence of microbial contamination (bacteria, fungus, virus, or mycoplasma) when tested 3-4 days before experimentation; CD73 and CD105 expression in more than 90% of the cells; and the absence of CD14, CD34, and CD45 in less than 3% of the cells, as assessed by flow cytometry.


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

Jang YO, Jun BG, Baik SK, Kim MY, Kwon SO - Clin Mol Hepatol (2015)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493357&req=5

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).
Mentions: The immunophenotypes of the BM-MSCs (CD14, CD34, CD45, CD73, and CD105) were analyzed prior to experimental use, and their differentiation potentials were determined (osteogenesis and adipogenesis; Fig. 1). For the immunophenotype analysis, BM-MSCs were stained with antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): CD14-FITC, CD34-FITC, CD45-FITC, CD73-PE, and CD105-PE (BD Biosciences, San Jose, CA, USA). Briefly, 5×105 cells were resuspended in 0.2 mL of phosphate-buffered saline (PBS) and incubated with FITC- or PE-conjugated antibodies for 20 min at room temperature. FITC- or PE-conjugated mouse IgGs were used as the control isotype at the same concentration as the specific primary antibodies. The fluorescence intensity of the cells was evaluated through flow cytometry (Epics XL; Beckman Coulter, Miami, FL, USA). Osteogenic differentiation was determined by first plating the cells at 2×104 cells/cm2 in six-well plates and then incubating them in the following osteogenic medium for 2-3 weeks: DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS, 10 mM β-glycerophosphate, 10-7 M dexamethasone, and 0.2 mM ascorbic acid (Sigma-Aldrich).20 The osteogenic differentiation was quantified from the release of p-nitrophenol from p-nitrophenyl phosphate by the enzyme alkaline phosphatase.21 For adipogenic differentiation, BM-MSCs were plated at 2×104 cells/cm2 in six-well plates and cultured for 1 week; then, differentiation was induced with adipogenic medium (10% FBS, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 µg/mL insulin, and 100 µM indomethacin in high-glucose DMEM) for an additional 3 weeks. The differentiated cells were fixed in 4% paraformaldehyde for 10 min and stained with fresh Oil Red-O solution (Sigma-Aldrich) to display lipid droplets (Fig. 1B). The criteria regarding the use of MSCs included viability greater than 80%; the absence of microbial contamination (bacteria, fungus, virus, or mycoplasma) when tested 3-4 days before experimentation; CD73 and CD105 expression in more than 90% of the cells; and the absence of CD14, CD34, and CD45 in less than 3% of the cells, as assessed by flow cytometry.

Bottom Line: Therapies involving bone-marrow-derived mesenchymal stem cells (BM-MSCs) have considerable potential in the management of hepatic disease.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.These results have demonstrated that BM-MSCs may exert an antifibrosis effect by modulating the function of activated HSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea. ; Cell Therapy and Tissue Engineering Center, Yonsei University Wonju College of Medicine, Wonju, Korea.

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.

No MeSH data available.


Related in: MedlinePlus