Characterization of adipose tissue-derived stromal cells of mice with nonalcoholic fatty liver disease and their use for liver repair.

INTRODUCTION: Freshly isolated uncultured adipose tissue-derived stromal cells (u-ADSCs), containing miscellaneous cells like the relatively abundant mesenchymal stem cells, are attractive for repair and regenerative therapy. However, the detailed characteristics and therapeutic efficacy of u-ADSCs obtained from disease-affected hosts are unknown. We compared the properties of u-ADSCs obtained from wild-type mice and from a mouse model of non-alcoholic steatohepatitis (NASH). METHODS: The NASH model was established by feeding C57BL/6J mice an atherogenic high-fat diet for 4 (NASH (4w)) or 12 weeks (NASH (12w)), followed by the isolation and characterization of u-ADSCs. Wild-type u-ADSCs or NASH-derived u-ADSCs were administered to mice with NASH cirrhosis, followed by analyses of hepatic inflammatory cells, antigen profiles, fibrosis, and gene expression. RESULTS: Wild-type u-ADSCs and NASH-derived u-ADSCs did not show marked differences in surface antigen profiles. In NASH (4w) u-ADSCs, but not NASH (12w) u-ADSCs, the frequencies of the leukocyte markers CD11b, CD45, and CD44 were elevated; furthermore, we observed an increase in the M1/M2 macrophage ratio only in NASH (12w) u-ADSCs. Only in NASH-4w u-ADSCs, the expression levels cell cycle-related genes were higher than those in u-ADSCs. Wild-type u-ADSCs administered to mice with NASH-related cirrhosis decreased the infiltration of CD11b+, F4/80+, and Gr-1+ inflammatory cells, ameliorated fibrosis, and had a restorative effect on liver tissues, as determined by gene expression profiles and the NAFLD activity score. The therapeutic effects of NASH (4w) u-ADSCs and NASH (12w) u-ADSCs on NASH-related cirrhosis were highly similar to the effect of wild-type u-ADSCs, including reductions in inflammation and fibrosis. CONCLUSIONS: NASH-derived u-ADSCs, similar to wild-type u-ADSCs, are applicable for reparative and regenerative therapy in mice with NASH.

Restorative effect of adipose tissue-derived stem cells on impaired hepatocytes through Notch signaling in non-alcoholic steatohepatitis mice.

Adipose tissue-derived stem cells (ADSCs) have been suggested as a novel treatment for non-alcoholic steatohepatitis (NASH); however, the mechanisms underlying their therapeutic effect remain poorly understood. In this study, we aimed to investigate the association of Notch signaling, which is crucial for cellular proliferation and differentiation in ADSC-mediated treatment of NASH. Flow cytometry analysis of ADSCs showed that they expressed the Notch ligands JAG1, DLL1, and DLL4. The expression of genes associated with the Notch signaling pathway was attenuated in hepatocytes of NASH model mice. We further observed ADSC-mediated activation of Notch signaling in these hepatocytes in addition to an increase in proliferating cell nuclear antigen+ cells and a decrease in TdT-mediated dUTP-biotin nick end labeling+ apoptotic cells. Co-culture of palmitic acid-induced steatotic hepatocytes and ADSCs resulted in the activation of Notch signaling and reduction of apoptosis of steatotic hepatocytes. Moreover, inhibition of Notch signaling by a γ-secretase inhibitor and knockdown of Notch ligands using siRNA attenuated the anti-apoptotic effect of co-cultured ADSCs in vitro. Our findings show that the Notch signaling pathway is involved in the inhibition of apoptosis and restoration of cellular proliferation of hepatocytes from NASH mice following ADSC treatment.

Combination of gemcitabine and anti-PD-1 antibody enhances the anticancer effect of M1 macrophages and the Th1 response in a murine model of pancreatic cancer liver metastasis.

BACKGROUND: Pancreatic ductular adenocarcinoma (PDAC) is among the most dreadful of malignancies, in part due to the lack of efficacious chemotherapy. Immune checkpoint inhibitors, including anti-programmed cell death 1 (anti-PD-1) antibodies, are novel promising forms of systemic immunotherapy. In the current study, we assessed whether gemcitabine (GEM) combined with anti-PD-1 antibody treatment was efficacious as immunochemotherapy for advanced PDAC using a murine model of liver metastasis. METHODS: The murine model of PDAC liver metastasis was established by intrasplenically injecting the murine pancreatic cancer cell line PAN02 into immunocompetent C57BL/6J mice. The mice were treated with an anti-PD-1 antibody, GEM, or a combination of GEM plus anti-PD-1 antibody, and compared with no treatment (control); liver metastases, immune cell infiltration, gene expression, immune cell response phenotypes, and overall survival were investigated. RESULTS: In the metastatic tumor tissues of mice treated with GEM plus anti-PD-1 antibody, we observed the increased infiltration of Th1 lymphocytes and M1 macrophages. Gene expression profile analysis of peripheral blood cells obtained from mice treated with GEM plus anti-PD-1 antibody clearly highlighted T cell and innate immune signaling pathways. Survival of PDAC liver metastasis mice was significantly prolonged by the combination therapy (median survival, 66 days) when compared with that of GEM alone treatment (median survival, 56 days). Expanded lymphocytes, which were isolated from the splenocytes of PDAC liver metastasis mice treated with GEM plus anti-PD-1 antibody, had an increased number of M1 macrophages. CONCLUSION: The combination of anti-PD-1 antibody immunotherapy with GEM was beneficial to treat a murine model of PDAC liver metastasis by enhancing the immune response mediated by Th1 lymphocytes and M1 macrophages and was associated with CD8+ T cells.