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. 2015 Apr 14;6(1):75.
doi: 10.1186/s13287-015-0072-7.

Lipopolysaccharide preconditioning of adipose-derived stem cells improves liver-regenerating activity of the secretome

Sang Chul Lee  1 , Hye Jin Jeong  2 , Sang Kuon Lee  3 , Say-June Kim  4
Affiliations

Lipopolysaccharide preconditioning of adipose-derived stem cells improves liver-regenerating activity of the secretome

Sang Chul Lee et al. Stem Cell Res Ther. .

Abstract

Introduction: Growing recognition of paracrine mechanisms in stem cell plasticity has resulted in considerable interest in stem cell-derived secretome. The aim of this study was to investigate the effects of lipopolysaccharide (LPS) preconditioning on the composition and hepatic regenerative activity of adipose-derived stem cell (ASC) secretome.

Methods: Conditioned medium (CM) and LPS-CM were obtained after culturing human ASCs without or with low-dose LPS (0.5 ng/mL) for 24 hours. Untreated and thioacetamide-treated mouse AML12 hepatocytes were incubated for 24 hours with the control medium, LPS (0.5 ng/mL), CM, and LPS-CM and then cell viabilities were compared. CM and LPS-CM were also intravenously administered to partially hepatectomized mice, and their effects on liver regeneration were assessed by using liver weight measurements, immunohistochemistry, and Western blotting.

Results: In the in vitro experiments, LPS preconditioning of ASCs enhanced the mRNA expression levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), hepatocyte growth factor, and vascular endothelial growth factor, which evoke inflammatory response or liver regeneration. LPS-CM significantly promoted thioacetamide-damaged AML12 cell viability compared with CM-incubated cells and the control cells (77%, 69%, and 65% P<0.05). In the in vivo experiment, LPS-CM infusion into the partially hepatectomized mice significantly reduced serum IL-6 and TNF-α levels compared with the other groups (P<0.05) on days 1 and 2 after partial hepatectomy. Moreover, LPS-CM infusion enhanced liver regeneration (based on the liver weight changes at day 7 after partial hepatectomy, 3.73% versus 3.22% in the CM group; P<0.05) and significantly reduced the elevated serum levels of aspartate transaminase and alanine transaminase (at day 1, P<0.05).

Conclusions: Our results suggest that LPS preconditioning effectively stimulates ASCs to produce the secretome beneficial to hepatic regeneration. Thus, optimizing ASC secretome profile by LPS preconditioning could be a promising approach to treat liver diseases by using stem cells.

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Figures

Figure 1
Figure 1
Effects of lipopolysaccharide (LPS) preconditioning of adipose tissue-derived stem cells (ASCs) on mRNA expression and secretion of pro-inflammatory cytokines. (A) ASCs were cultured in serum-free low-glucose Dulbecco’s modified Eagle’s medium with or without LPS in low concentration (0.5 ng/mL) for 24 hours and analyzed for mRNA expression of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) by quantitative polymerase chain reaction. (B) Conditioned media (CM and LPS-CM) were collected, concentrated 25-fold, and analyzed for IL-6 concentration by enzyme-linked immunosorbent assay. Values represent mean ± standard deviation of four independent experiments (P <0.05). *P <0.05. CM, conditioned medium; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 2
Figure 2
Effects of conditioned medium (CM) and lipopolysaccharide (LPS)-CM on the viability of normal and hepatotoxic agent-treated mouse hepatocytes. (A) Untreated and 50 mM thioacetamide (TAA)-treated and TAA + TLR4 inhibitor (TAK-242) AML12 cells were incubated for 24 hours with the control medium, LPS (0.5 ng/mL), CM, and LPS-CM and cell viabilities were determined by using the Ez-Cytox cell viability assay. Values represent mean ± standard deviation of four independent experiments (P <0.05). (B) The expressions of phospho-signal transducer and activator of transcription 3 (p-STAT3) and STAT3 in AML12 cells treated as above were analyzed by Western blotting. *P <0.05. TLR4, Toll-like receptor 4.
Figure 3
Figure 3
Serum concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in partially hepatectomized mice treated with adipose tissue-derived stem cell-conditioned media (CM). Mice were intravenously injected with low-glucose Dulbecco’s modified Eagle’s medium (control), lipopolysaccharide (LPS) (0.5 ng/mL), CM, or LPS-CM 1 hour after partial hepatectomy (PH) and tested for TNF-α (A) and IL-6 (B) serum levels at indicated times after PH (P <0.05). In each group, five mice were included per time interval (all 60 mice). Values represent mean ± standard deviation. *P <0.05.
Figure 4
Figure 4
Effects of conditioned medium (CM) and lipopolysaccharide (LPS)-CM on liver regeneration in partially hepatectomized mice. Mice were intravenously injected with low-glucose Dulbecco’s modified Eagle’s medium (control), LPS (0.5 ng/mL), CM, or LPS-CM 1 hour after partial hepatectomy (PH). (A) Representative micrographs show liver sections stained with anti-Ki67 antibody 24 hours after PH (magnification, 400×). (B) The percentage of Ki67-positive cells in the liver on days 1, 2, and 3 after PH (P <0.05). (C) Western blotting of liver lysates showing that the LPS-CM group had the highest expression of PCNA and p-STAT3 on day 2 after PH. (D) Liver regeneration rate (percentage) based on the ratio (percentage) of liver weight to body weight (LW/BW) on day 7 after PH; the highest regeneration was observed in the LPS-CM group (P <0.05). *P <0.05. PCNA, proliferating cell nuclear antigen; p-STAT3, phospho-signal transducer and activator of transcription 3; STAT3, signal transducer and activator of transcription 3.
Figure 5
Figure 5
Effect of conditioned medium (CM) and lipopolysaccharide (LPS)-CM on serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) in partially hepatectomized mice. Mice were intravenously injected with low-glucose Dulbecco’s modified Eagle’s medium (control), LPS (0.5 ng/mL), CM, or LPS-CM 1 hour after partial hepatectomy. At day 1 after partial hepatectomy, the LPS-CM-treated mice had significantly lower AST (A) and ALT (B) levels compared with the other groups (P <0.05). *P <0.05.
Figure 6
Figure 6
Proposed mechanisms by which lipopolysaccharide-conditioned medium (LPS-CM) induces liver regeneration in partially hepatectomized mice. It can be postulated that the LPS-preconditioned secretome releases a variety of mediators-including interleukin-6 (IL-6), tumor necrosis factor-alpha, hepatocyte growth factor, and vascular endothelial growth factor—from adipose tissue-derived stem cells (ASCs) by way of intracellular nuclear factor-kappa-B (NF-κB) activation. These mediators promote hepatocyte proliferation by way of various signaling pathways, including IL-6/STAT3 signaling. JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; TLR4, Toll-like receptor 4.
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