Generation of induced secretome from adipose-derived stem cells specialized for disease-specific treatment: An experimental mouse model

Figure 6 Validation of disease specificity and analysis of secretome components. A: Western blot analysis showing effectiveness of the TAA-induced secretome (isecretome) in partially hepatectomized mice. TAA-induced conditioned media (iCM) infusion was inferior to CM infusion in partially hepatectomized mice, considering the lower expression of proliferation markers (p-ERK and PCNA), and an anti-apoptotic marker (Mcl-1) and higher expression of a proapoptotic marker (Bax) following TAA-iCM infusion; B: Heat map generated from label-free liquid chromatography–mass spectrometry for quantitative proteomics reflecting protein expression values of the secretome, TAA-isecretome, and HBx-isecretome. Samples are arranged in columns and proteins in rows. Red shades indicate increased expression in samples compared to in control samples; green shades indicate reduced expression; black indicates median expression. Each sample for liquid chromatography–mass spectrometry was pooled from three samples of the secretome. The components and concentration of various essential proteins varied widely between iCM and HBx-iCM, validating their specificity. Values are presented as mean ± SD of three independent experiments; C: Gene ontology enrichment analysis of the ten proteins that had been exclusively identified in the TAA-isecretome. Gene ontology enrichment analysis identified the 19 enriched biological networks of the 10 proteins. Of the 19 enriched biological networks, two of the most prominent biological processes were the response to reactive oxygen species and cell redox homeostasis, all of which were related to antioxidant activity; D: Peroxiredoxin-1 (Prdx-1) inhibition test. Prdx-1 is one of the potent antioxidant proteins and was found to be one of ten proteins that had been exclusively identified in the TAA-isecretome. We performed Prdx-1 inhibition test for the determination of the role of Prdx-1 in the hepatic reparative process. Herein, liver regenerative capacity was estimated by the expression of a hepatoproliferative marker (p-ERK). When treated with the TAA-iCM, AML2 hepatocytes showed increased expression of p-ERK as well as Prdx-1 compared with AML2 cells treated with the control CM (P < 0.05). Subsequently, we compared the efficacy of the TAA-iCM after pre-treatment of TAK242, a TLR4 inhibitor. Pre-treatment of TAK242 did not only inhibit the expression of Prdx-1 but also inhibited the expression of p-ERK, the hepatoproliferative marker, suggesting the hepatoprotective role of Prdx-1 in the liver. ^cP < 0.05 vs control (saline); ^eP < 0.05 between CM and iCM; ^gP < 0.05 between iCMs with or without TAK242. BIM: Bcl-2-like protein 11; iCM: Isecretome group of which components were the TAA-induced secretome; HBx-iCM: Isecretome group of which components were the HBx-induced secretome; Mcl-1: Myeloid cell leukemia 1; CM: Normal conditioned media group of which components were the naïve secretome; PCNA: Proliferating cell nuclear antigen; Prdx-1: Peroxiredoxin-1.