Etanercept-synthesizing adipose-derived stem cell secretome: A promising therapeutic option for inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract, with tumor necrosis factor (TNF)-α playing a key role in its pathogenesis. Etanercept, a decoy receptor for TNF, is used to treat inflammatory conditions. The secretome derived from adipose-derived stem cells (ASCs) has anti-inflammatory effects, making it a promising therapeutic option for IBD. AIM: To investigate the anti-inflammatory effects of the secretome obtained from ASCs synthesizing etanercept on colon cells and in a dextran sulfate sodium (DSS)-induced IBD mouse model. METHODS: ASCs were transfected with etanercept-encoding mini-circle plasmids to create etanercept-producing cells. The secretory material from these cells was then tested for anti-inflammatory effects both in vitro and in a DSS-induced IBD mouse model. RESULTS: This study revealed promising results indicating that the group treated with the secretome derived from etanercept-synthesizing ASCs [Etanercept-Secretome (Et-Sec) group] had significantly lower expression levels of inflammatory mediators, such as interleukin-6, Monocyte Chemoattractant Protein-1, and TNF-α, when compared to the control secretome (Ct-Sec). Moreover, the Et-Sec group exhibited a marked therapeutic effect in terms of preserving the architecture of intestinal tissue compared to the Ct-Sec. CONCLUSION: These results suggest that the secretome derived from ASCs that synthesize etanercept has potential as a therapeutic agent for the treatment of IBD, potentially enhancing treatment efficacy by merging the anti-inflammatory qualities of the ASC secretome with etanercept's targeted approach to better address the multifaceted pathophysiology of IBD.

Cutting-Edge HEK293T Protein-Integrated Lipid Nanostructures: Boosting Biocompatibility and Efficacy.

Recently, artificial exosomes have been developed to overcome the challenges of natural exosomes, such as production scalability and stability. In the production of artificial exosomes, the incorporation of membrane proteins into lipid nanostructures is emerging as a notable approach for enhancing biocompatibility and treatment efficacy. This study focuses on incorporating HEK293T cell-derived membrane proteins into liposomes to create membrane-protein-bound liposomes (MPLCs), with the goal of improving their effectiveness as anticancer therapeutics. MPLCs were generated by combining two key elements: lipid components that are identical to those in conventional liposomes (CLs) and membrane protein components uniquely derived from HEK293T cells. An extensive comparison of CLs and MPLCs was conducted across multiple in vitro and in vivo cancer models, employing advanced techniques such as cryo-TEM (tramsmission electron microscopy) imaging and FT-IR (fourier transform infrared spectroscopy). MPLCs displayed superior membrane fusion capabilities in cancer cell lines, with significantly higher cellular uptake. Additionally, MPLCs maintained their morphology and size better than CLs when exposed to FBS (fetal bovine serum), suggesting enhanced serum stability. In a xenograft mouse model using HeLa and ASPC cancer cells, intravenous administration of MPLCs MPLCs accumulated more in tumor tissues, highlighting their potential for targeted cancer therapy. Overall, these results indicate that MPLCs have superior tumor-targeting properties, possibly attributable to their membrane protein composition, offering promising prospects for enhancing drug delivery efficiency in cancer treatments. This research could offer new clinical application opportunities, as it uses MPLCs with membrane proteins from HEK293T cells, which are known for their efficient production and compatibility with GMP (good manufacturing practice) standards.

Pioneering PGC-1α-boosted secretome: a novel approach to combating liver fibrosis.

Purpose: Liver fibrosis is a critical health issue with limited treatment options. This study investigates the potential of PGC-Sec, a secretome derived from peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-overexpressing adipose-derived stem cells (ASCs), as a novel therapeutic strategy for liver fibrosis. Methods: Upon achieving a cellular confluence of 70%-80%, ASCs were transfected with pcDNA-PGC-1α. PGC-Sec, obtained through concentration of conditioned media using ultrafiltration units with a 3-kDa cutoff, was assessed through in vitro assays and in vitro mouse models. Results: In vitro, PGC-Sec significantly reduced LX2 human hepatic stellate cell proliferation and mitigated mitochondrial oxidative stress compared to the control-secretome. In an in vivo mouse model, PGC-Sec treatment led to notable reductions in hepatic enzyme activity, serum proinflammatory cytokine concentrations, and fibrosis-related marker expression. Histological analysis demonstrated improved liver histology and reduced fibrosis severity in PGC-Sec-treated mice. Immunohistochemical staining confirmed enhanced expression of PGC-1α, optic atrophy 1 (a mitochondrial function marker), and peroxisome proliferator-activated receptor alpha (an antifibrogenic marker) in the PGC-Sec-treated group, along with reduced collagen type 1A expression (a profibrogenic marker). Conclusion: These findings highlight the therapeutic potential of PGC-Sec in combating liver fibrosis by enhancing mitochondrial biogenesis and function, and promoting antifibrotic processes. PGC-Sec holds promise as a novel treatment strategy for liver fibrosis.

The secretome obtained under hypoxic preconditioning from human adipose-derived stem cells exerts promoted anti-apoptotic potentials through upregulated autophagic process.

BACKGROUND: Hypoxic preconditioning (HP) is a stem cell preconditioning modality designed to augment the therapeutic effects of mesenchymal stem cells (MSCs). Although autophagy is expected to play a role in HP, very little is known regarding the relationship between HP and autophagy. METHODS AND RESULTS: The adipose-derived stem cell (ASC)-secretome obtained under normoxia (NCM) and ASC-secretome obtained under HP (HCM) were obtained by culturing ASCs for 24 h under normoxic (21% partial pressure of O2) and hypoxic (1% partial pressure of O2) conditions, respectively. Subsequently, to determine the in vivo effects of HCM, each secretome was injected into 70% partially hepatectomized mice, and liver specimens were obtained. HCM significantly reduced the apoptosis of thioacetamide-treated AML12 hepatocytes and promoted the autophagic processes of the cells (P < 0.05). Autophagy blockage by either bafilomycin A1 or ATG5 siRNA significantly abrogated the anti-apoptotic effect of HCM (P < 0.05), demonstrating that HCM exerts its anti-apoptotic effect by promoting autophagy. The effect of HCM - reduction of cell apoptosis and promotion of autophagic process - was also demonstrated in a mouse model. CONCLUSIONS: HP appears to induce ASCs to release a secretome with enhanced anti-apoptotic effects by promoting the autophagic process of ASCs.

Robotic surgery enables safe and comfortable single-incision cholecystectomy: A comparison of robotic and laparoscopic approaches for single-incision surgery.

BACKGROUND: Although single-incision robotic cholecystectomy (SIRC) overcomes various limitations of single-incision laparoscopic cholecystectomy (SILC), it is associated with high cost. In this study, we intended to investigate if SIRC is recommendable and advantageous to patients despite its high cost. MATERIALS AND METHODS: We prospectively collected and analysed data of patients who had undergone either SILC (n = 25) or SIRC (n = 50) for benign gallbladder diseases, with identical inclusion criteria, between November 2017 and February 2019. RESULTS: SILC and SIRC showed similar operative outcomes in terms of intra- and post-operative complications and verbal numerical rating scale (VNRS) for pain. However, the SIRC group exhibited significantly longer operation time than the SILC group (83.2 ± 32.6 vs. 66.4 ± 32.8, P = 0.002). The SIRC group also showed longer hospital stay (2.4 ± 0.7 vs. 2.2 ± 0.6, P = 0.053). Although the SILC and SIRC groups showed no significant difference in VNRS, the SIRC group required a higher amount (126.0 ± 88.8 mg vs. 87.5 ± 79.7 mg, P = 0.063) and frequency (3.0 ± 2.1 vs. 2.0 ± 1.8, P = 0.033) of intravenous opioid analgesic administration. During surgery, the critical view of safety (CVS), the prerequisite for safe cholecystectomy, was identified in only 24% (n = 6) of patients undergoing SILC and in 100% (n = 50) of patients undergoing SIRC (P < 0.05). CONCLUSION: We conclude that although SILC and SIRC have similar operative outcomes, SIRC is advantageous over SILC because of its potential to markedly enhance the safety of patients by proficiently acquiring CVS.

Potentiation of the Anticancer Effects by Combining Docetaxel with Ku-0063794 against Triple-Negative Breast Cancer Cells.

PURPOSE: mTORC1 and mTORC2 inhibition by Ku-0063794 could confer profound anticancer effects against cancer cells because it eliminates feedback activation of Akt. Herein, we aimed to determine anticancer effects of docetaxel and Ku-0063794, individually or in combination, against breast cancer cells, especially triple-negative breast cancer (TNBC) cells. MATERIALS AND METHODS: MCF-7 breast cancer and MDA-MB-231 TNBC cell lines for in vitro studies and mouse xenograft model for in vivo studies were used to investigate the effect of docetaxel, Ku-0063794, or their combination. RESULTS: In the in vitro experiments, combination therapy synergistically reduced cell viability and induced higher apoptotic cell death in breast cancer cells than the individual monotherapies (p < 0.05). Western blot analysis and flow cytometric analysis showed that the combination therapy induced higher apoptotic cell death than the individual monotherapies (p < 0.05). In the in vivo experiment, docetaxel and Ku-0063794 combination therapy reduced the growth of MDA-MB-231 cells xenografted in the nude mice better than in the individual monotherapies (p < 0.05). Immunohistochemistry showed that the combination therapy induced the highest expression of cleaved caspase-3 and the lowest expression of Bcl-xL in the MDA-MB-231 cells xenografted in the nude mice (p < 0.05). Western blot analysis and immunofluorescence, incorporating both in vitro and in vivo experiments, consistently validated that unlike individual monotherapies, docetaxel and Ku-0063794 combination therapy significantly inhibited epithelial-mesenchymal transition (EMT) and autophagy (p < 0.05). CONCLUSION: These data suggest that docetaxel and Ku-0063794 combination therapy has higher anticancer activities over individual monotherapies against MDA-MB-231 TNBC cells through a greater inhibition of autophagy and EMT.

A novel strategy to promote liver regeneration: utilization of secretome obtained from survivin-overexpressing adipose-derived stem cells.

PURPOSE: Survivin is a typical antiapoptotic protein. It is copiously expressed during human fetal development but is infrequently present in adult tissues. In this experiment, we researched the treatment effect of the secretome that adipose-derived stem cells (ASCs) transfected with survivin. METHODS: First of all, we generated survivin-overexpressing ASCs transfected with a plasmid comprising a gene encoding survivin. The secreted substances released from survivin-overexpressing ASCs (survivin-secretome) were collected, and were determined their in vitro and in vivo therapeutic potential, especially in the model of liver impairment. RESULTS: In vitro, the survivin-secretome significantly increased cell viability and promoted the expression of proliferation-related markers (proliferating cell nuclear antigen [PCNA], phospho-signal transducer and activator of transcription 3 (p-STAT3), hepatocyte growth factor [HGF], vascular endothelial growth factor [VEGF]) and anti-apoptosis-related markers (myeloid cell leukemia-1 [Mcl-1] and survivin) (P < 0.05). In vivo using 70% hepatectomy mice, the survivin-secretome group exhibited the lowest serum levels of interleukin-6, tumor necrosis factor-α (P < 0.05). The serum levels of liver transaminases (alanine aminotransferase and aspartate aminotransferase) were also the lowest in the survivin-secretome group (P < 0.05). The survivin-secretome group also exhibited the highest liver regeneration on the 7th day after 70% partial hepatectomy (P < 0.05). In the subsequent liver specimen analysis, the specimens of survivin-secretome exhibited the highest expression of p-STAT3, HGF, VEGF, PCNA, and Mcl-1 and the lowest expression of bcl-2-like protein 4 (P < 0.05). CONCLUSION: Taken together, secretome secreted by survivin-overexpressing ASCs could be an effective way to improve liver regeneration and repair for liver injury treatment.

Combining Everolimus and Ku0063794 Promotes Apoptosis of Hepatocellular Carcinoma Cells via Reduced Autophagy Resulting from Diminished Expression of miR-4790-3p.

It is challenging to overcome the low response rate of everolimus in the treatment of patients with hepatocellular carcinoma (HCC). To overcome this challenge, we combined everolimus with Ku0063794, the inhibitor of mTORC1 and mTORC2, to achieve higher anticancer effects. However, the precise mechanism for the synergistic effects is not clearly understood yet. To achieve this aim, the miRNAs were selected that showed the most significant variation in expression according to the mono- and combination therapy of everolimus and Ku0063794. Subsequently, the roles of specific miRNAs were determined in the processes of the treatment modalities. Compared to individual monotherapies, the combination therapy significantly reduced viability, increased apoptosis, and reduced autophagy in HepG2 cells. The combination therapy led to significantly lower expression of miR-4790-3p and higher expression of zinc finger protein225 (ZNF225)-the predicted target of miR-4790-3p. The functional study of miR-4790-3p and ZNF225 revealed that regarding autophagy, miR-4790-3p promoted it, while ZNF225 inhibited it. In addition, regarding apoptosis, miR-4790-3p inhibited it, while ZNF225 promoted it. It was also found that HCC tissues were characterized by higher expression of miR-4790-3p and lower expression of ZNF225; HCC tissues were also characterized by higher autophagic flux. We, thus, conclude that the potentiated anticancer effect of the everolimus and Ku0063794 combination therapy is strongly associated with reduced autophagy resulting from diminished expression of miR-4790-3p, as well as higher expression of ZNF225.

Harnessing adipose­derived stem cells to release specialized secretome for the treatment of hepatitis B.

Mesenchymal stem cells (MSCs) have the function of repairing damaged tissue, which is known to be mediated by the secretome, the collection of secretory materials shed from MSCs. Adjusting the culture conditions of MSCs can lead to a significant difference in the composition of the secretome. It was hypothesized that pre­sensitization of MSCs with specific disease­causing agents could harness MSCs to release the therapeutic materials specialized for the disease. To validate this hypothesis, the present study aimed to generate a 'disease­specific secretome' for hepatitis caused by hepatitis B virus using hepatitis BX antigen (HBx) as a disease­causing material. Secretary materials (HBx­IS) were collected following the stimulation of adipose­derived stem cells (ASCs) with 100­fold diluted culture media of AML12 hepatocytes that had been transfected with pcDNA­HBx for 24 h. An animal model of hepatitis B was generated by injecting HBx into mice, and the mice were subsequently intravenously administered a control secretome (CS) or HBx­IS. Compared with the CS injection, the HBx­IS injection significantly reduced the serum levels of interleukin­6 and tumor necrosis factor­α (pro­inflammatory cytokines). Western blot analysis and immunohistochemistry of the liver specimens revealed that the HBx­IS injection led to a higher expression of liver regeneration­related markers, including hepatocyte growth factor and proliferating cell nuclear antigen, a lower expression of pro­apoptotic markers, such as cleaved caspase 3 and Bim in mouse livers, and a lower expression of pro­inflammatory markers (F4/80 and CD68) compared to the CS injection. HBx­IS exhibited higher liver regenerative, anti­inflammatory and anti­apoptotic properties, particularly in the mouse model of hepatitis B compared to CS. This suggests that the secretome obtained by stimulating ASCs with disease­causing agents may have a more prominent therapeutic effect on the specific disease than the naïve secretome.

Application of self-assembly peptides targeting the mitochondria as a novel treatment for sorafenib-resistant hepatocellular carcinoma cells.

Currently, there is no appropriate treatment option for patients with sorafenib-resistant hepatocellular carcinoma (HCC). Meanwhile, pronounced anticancer activities of newly-developed mitochondria-accumulating self-assembly peptides (Mito-FF) have been demonstrated. This study intended to determine the anticancer effects of Mito-FF against sorafenib-resistant Huh7 (Huh7-R) cells. Compared to sorafenib, Mito-FF led to the generation of relatively higher amounts of mitochondrial reactive oxygen species (ROS) as well as the greater reduction in the expression of antioxidant enzymes (P < 0.05). Mito-FF was found to significantly promote cell apoptosis while inhibiting cell proliferation of Huh7-R cells. Mito-FF also reduces the expression of antioxidant enzymes while significantly increasing mitochondrial ROS in Huh7-R cells. The pro-apoptotic effect of Mito-FFs for Huh7-R cells is possibly caused by their up-regulation of mitochondrial ROS, which is caused by the destruction of the mitochondria of HCC cells.

PGC-1α is downregulated in a mouse model of obstructive cholestasis but not in a model of liver fibrosis.

Several studies have indicated that cholestatic liver damage involves mitochondria dysfunction. However, the precise mechanism by which hydrophobic bile salts cause mitochondrial dysfunction is not clear. In this study, we intended to determine the pathogenesis of cholestatic liver injury associated with peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α). A mouse model of cholestatic liver disease was generated by surgical ligation of the bile duct (BDL), and a mouse model of fibrosis was developed through serial administration of thioacetamide. After obtaining liver specimens on scheduled days, we compared the expression of the antioxidant enzymes (superoxide dismutase 2 [SOD2], catalase, and glutathione peroxidase-1[GPx-1]) and PGC-1α in livers from mice with fibrosis and cholestasis using western blotting, immunohistochemistry, and immunofluorescence. We found that cholestatic livers exhibit lower expression of antioxidant enzymes, such as SOD2, catalase, and PGC-1α. In contrast, fibrotic livers exhibit higher expression of antioxidant enzymes and PGC-1α. In addition, cholestatic livers exhibited significantly lower expression of pro-apoptotic markers (Bax) as compared to fibrotic livers. It is well known that overexpression of PGC-1α increases mitochondrial antioxidant enzyme expression, and vice versa. Thus, we concluded that obstructive cholestasis decreases expression of PGC-1α, which may lead to decreased expression of mitochondrial antioxidant enzymes, thereby rendering mice with cholestatic livers vulnerable to ROS-induced cell death.

Novel Therapeutic Application of Self-Assembly Peptides Targeting the Mitochondria in In Vitro and In Vivo Experimental Models of Gastric Cancer.

Here, we provide the possibility of a novel chemotherapeutic agent against gastric cancer cells, comprising the combination of 5-fluorouracil (5-FU) and a mitochondria-targeting self-assembly peptide, which is a phenylalanine dipeptide with triphenyl phosphonium (Mito-FF). The anticancer effects and mechanisms of 5-FU and Mito-FF, individually or in combination, were compared through both in vitro and in vivo models of gastric cancer. Our experiments consistently demonstrated that the 5-FU and Mito-FF combination therapy was superior to monotherapy with either, as manifested by both higher reduction of proliferation as well as an induction of apoptotic cell death. Interestingly, we found that combining 5-FU with Mito-FF leads to a significant increase of reactive oxygen species (ROS) and reduction of antioxidant enzymes in gastric cancer cells. Moreover, the inhibition of ROS abrogated the pro-apoptotic effects of combination therapy, suggesting that enhanced oxidative stress could be the principal mechanism of the action of combination therapy. We conclude that the combination of 5-FU and Mito-FF exerts potent antineoplastic activity against gastric cancer cells, primarily by promoting ROS generation and suppressing the activities of antioxidant enzymes.

Serum level of visfatin can reflect the severity of inflammation in patients with acute cholecystitis.

PURPOSE: Visfatin is a key cytokine released from the pe ripheral blood mononuclear cells (PBMCs) as well as adipose tissue, and it is involved in immune response as well as inflammation. In this study, we investigated whether the serum visfatin level could be a prognostic factor for predicting the severity of inflammation in patients with acute cholecystitis. METHODS: We examined the blood samples and gallbladder specimens from patients who underwent laparoscopic cholecystectomy for either acute (n = 18) or chronic cholecystitis (n = 18). We determined the visfatin levels of these samples using various procedures such as real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, and immunohistochemistry. RESULTS: The patients with acute cholecystitis exhibited higher mRNA expression of visfatin in PBMCs, higher serum levels of visfatin, and increased protein expression of visfatin in the gallbladder specimens than in patients with chronic cholecystitis. In the in vitro model of acute cholecystitis, the mRNA expression of visfatin showed the fastest increase among the other pro-inflammatory mediators studied, including interleukin (IL)-10, tumor necrosis factor-α, IL-6, intracellular adhesion molecule-1, and ascular cell adhesion molecule-1. Inhibition of visfatin using siRNA abrogated the inhibitory effects of lipopolysaccharide (LPS) on the expression of ABCG1 in GBECs, suggesting that visfatin is significantly involved in the LPS-driven suppression of ABCG1. CONCLUSION: Taken together, we concluded that visfatin is a pro-inflammatory mediators that is upregulated during acute cholecystitis and is expected to be increased within a short time after inflammation. Therefore, measuring the serum level of visfatin would be helpful in predicting the inflammatory severity in the patients with acute cholecystitis.

A Novel Way of Preventing Postoperative Pancreatic Fistula by Directly Injecting Profibrogenic Materials into the Pancreatic Parenchyma.

This paper aims to validate if intrapancreatic injection of penicillin G can enhance hardness and suture holding capacity (SHC) of the pancreas through prompting the fibrosis process. Soft pancreatic texture is constantly mentioned as one of the most contributory predictors of postoperative pancreatic fistula (POPF). Soft pancreas has poor SHC and higher incidence of parenchymal tearing, frequently leading to POPF. From a library of 114 antibiotic compounds, we identified that penicillin G substantially enhanced pancreatic hardness and SHC in experimental mice. Specifically, we injected penicillin G directly into the pancreas. On determined dates, we measured the pancreatic hardness and SHC, respectively, and performed molecular and histological examinations for estimation of the degree of fibrosis. The intrapancreatic injection of penicillin G activated human pancreatic stellate cells (HPSCs) to produce various fibrotic materials such as transforming growth factor-ß1 (TGF-ß1) and metalloproteinases-2. The pancreatic hardness and SHC were increased to the maximum at the second day after injection and then it gradually subsided demonstrating its reversibility. Pretreatment of mice with SB431542, an inhibitor of the TGF-ß1 receptor, before injecting penicillin G intrapancreatically, significantly abrogated the increase of both pancreatic hardness and SHC caused by penicillin G. This suggested that penicillin G promotes pancreatic fibrosis through the TGF-ß1 signaling pathway. Intrapancreatic injection of penicillin G promotes pancreatic hardness and SHC by enhancing pancreatic fibrosis. We thus think that penicillin G could be utilized to prevent and minimize POPF, after validating its actual effectiveness and safety by further studies.

A novel antifibrotic strategy utilizing conditioned media obtained from miR-150-transfected adipose-derived stem cells: validation of an animal model of liver fibrosis.

The limitations of stem cells have led researchers to investigate the secretome, which is the secretory materials in stem cells, since the principal mechanism of action of stem cells is mediated by the secretome. In this study, we determined the antifibrotic potential of the secretome released from miR-150-transfected adipose-derived stromal cells (ASCs). The secretome released from ASCs that were transfected with antifibrotic miR-150 was obtained (referred to as the miR-150 secretome). To validate the antifibrotic effects of the miR-150 secretome, we generated in vitro and in vivo models of liver fibrosis by treating human hepatic stellate cells (LX2 cells) with thioacetamide (TAA) and subcutaneous injection of TAA into mice, respectively. In the in vitro model, more significant reductions in the expression of fibrosis-related markers, such as TGFß, Col1A1, and α-SMA, were observed by using the miR-150 secretome than the control secretome, specifically in TAA-treated LX2 cells. In the in vivo model, infusion of the miR-150 secretome into mice with liver fibrosis abrogated the increase in serum levels of systemic inflammatory cytokines, such as IL-6 and TNF-α, and induced increased expression of antifibrotic, proliferation, and antioxidant activity markers in the liver. Our in vitro and in vivo experiments indicate that the miR-150 secretome is superior to the naive secretome in terms of ameliorating liver fibrosis, minimizing systemic inflammatory responses, and promoting antioxidant enzyme expression. Therefore, we conclude that miR-150 transfection into ASCs has the potential to induce the release of secretory materials with enhanced antifibrotic, proliferative, and antioxidant properties.

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

BACKGROUND: Recently, the exclusive use of mesenchymal stem cell (MSC)-secreted molecules, named as the secretome, have been evaluated for overcoming the limitations of cell-based therapy while maintaining its advantages. AIM: To improve cell-free therapy by adding disease-specificity through stimulation of MSCs using disease-causing materials. METHODS: We collected the secretory materials (named as inducers) released from AML12 hepatocytes that had been pretreated with thioacetamide (TAA) and generated the TAA-induced secretome (TAA-isecretome) after stimulating adipose-derived stem cells with the inducers. The TAA-isecretome was intravenously administered to mice with TAA-induced hepatic failure and those with partial hepatectomy. RESULTS: TAA-isecretome infusion showed higher therapeutic potential in terms of (1) restoring disorganized hepatic tissue to normal tissue; (2) inhibiting proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α); and (3) reducing abnormally elevated liver enzymes (aspartate aminotransferase and alanine aminotransferase) compared to the naïve secretome infusion in mice with TAA-induced hepatic failure. However, the TAA-isecretome showed inferior therapeutic potential for restoring hepatic function in partially hepatectomized mice. Proteomic analysis of TAA-isecretome identified that antioxidant processes were the most predominant enriched biological networks of the proteins exclusively identified in the TAA-isecretome. In addition, peroxiredoxin-1, a potent antioxidant protein, was found to be one of representative components of TAA-isecretome and played a central role in the protection of TAA-induced hepatic injury. CONCLUSION: Appropriate stimulation of adipose-derived stem cells with TAA led to the production of a secretome enriched with proteins, especially peroxiredoxin-1, with higher antioxidant activity. Our results suggest that appropriate stimulation of MSCs with pathogenic agents can lead to the production of a secretome specialized for protecting against the pathogen. This approach is expected to open a new way of developing various specific therapeutics based on the high plasticity and responsiveness of MSCs.

A Novel Hepatic Anti-Fibrotic Strategy Utilizing the Secretome Released from Etanercept-Synthesizing Adipose-Derived Stem Cells.

Tumor necrosis factor-α (TNF-α)-driven inflammatory reaction plays a crucial role in the initiation of liver fibrosis. We herein attempted to design genetically engineered adipose-derived stem cells (ASCs) producing etanercept (a potent TNF-α inhibitor), and to determine the anti-fibrotic potential of the secretome released from the etanercept-synthesizing ASCs (etanercept-secretome). First, we generated the etanercept-synthesizing ASCs by transfecting the ASCs with mini-circle plasmids containing the gene insert encoding for etanercept. We subsequently collected the secretory material released from the etanercept-synthesizing ASCs and determined its anti-fibrotic effects both in vitro (in thioacetamide [TAA]-treated AML12 and LX2 cells) and in vivo (in TAA-treated mice) models of liver fibrosis. We observed that while etanercept-secretome increased the viability of the TAA-treated AML12 hepatocytes (p = 0.021), it significantly decreased the viability of the TAA-treated LX2 HSCs (p = 0.021). In the liver of mice with liver fibrosis, intravenous administration of the etanercept-secretome induced significant reduction in the expression of both fibrosis-related and inflammation-related markers compared to the control group (all Ps < 0.05). The etanercept-secretome group also showed significantly lower serum levels of liver enzymes as well as pro-inflammatory cytokines, such as TNF-α (p = 0.020) and IL-6 (p = 0.021). Histological examination of the liver showed the highest reduction in the degree of fibrosis in the entanercept-secretome group (p = 0.006). Our results suggest that the administration of etanercept-secretome improves liver fibrosis by inhibiting TNF-α-driven inflammation in the mice with liver fibrosis. Thus, blocking TNF-α-driven inflammation at the appropriate stage of liver fibrosis could be an efficient strategy to prevent fibrosis.

Enhanced Therapeutic Potential of the Secretome Released from Adipose-Derived Stem Cells by PGC-1α-Driven Upregulation of Mitochondrial Proliferation.

Peroxisome proliferator activated receptor λ coactivator 1α (PGC-1α) is a potent regulator of mitochondrial biogenesis and energy metabolism. In this study, we investigated the therapeutic potential of the secretome released from the adipose-derived stem cells (ASCs) transfected with PGC-1α (PGC-secretome). We first generated PGC-1α-overexpressing ASCs by transfecting ASCs with the plasmids harboring the gene encoding PGC-1α. Secretory materials released from PGC-1α-overexpressing ASCs were collected and their therapeutic potential was determined using in vitro (thioacetamide (TAA)-treated AML12 cells) and in vivo (70% partial hepatectomized mice) models of liver injury. In the TAA-treated AML12 cells, the PGC-secretome significantly increased cell viability, promoted expression of proliferation-related markers, such as PCNA and p-STAT, and significantly reduced the levels of reactive oxygen species (ROS). In the mice, PGC-secretome injections significantly increased liver tissue expression of proliferation-related markers more than normal secretome injections did (p < 0.05). We demonstrated that the PGC-secretome does not only have higher antioxidant and anti-inflammatory properties, but also has the potential of significantly enhancing liver regeneration in both in vivo and in vitro models of liver injury. Thus, reinforcing the mitochondrial antioxidant potential by transfecting ASCs with PGC-1α could be one of the effective strategies to enhance the therapeutic potential of ASCs.

Ameliorating liver fibrosis in an animal model using the secretome released from miR-122-transfected adipose-derived stem cells.

BACKGROUND: Recently, the exclusive use of mesenchymal stem cell (MSC)-secreted molecules, called secretome, rather than cells, has been evaluated for overcoming the limitations of cell-based therapy, while maintaining its advantages. However, the use of naïve secretome may not fully satisfy the specificity of each disease. Therefore, it appears to be more advantageous to use the functionally reinforced secretome through a series of processes involving physico-chemical adjustments or genetic manipulation rather than to the use naïve secretome. AIM: To determine the therapeutic potential of the secretome released from miR-122-transfected adipose-derived stromal cells (ASCs). METHODS: We collected secretory materials released from ASCs that had been transfected with antifibrotic miR-122 (MCM) and compared their antifibrotic effects with those of the naïve secretome (CM). MCM and CM were intravenously administered to the mouse model of thioacetamide-induced liver fibrosis, and their therapeutic potentials were compared. RESULTS: MCM infusion provided higher therapeutic potential in terms of: (A) Reducing collagen content in the liver; (B) Inhibiting proinflammatory cytokines; and (C) Reducing abnormally elevated liver enzymes than the infusion of the naïve secretome. The proteomic analysis of MCM also indicated that the contents of antifibrotic proteins were significantly elevated compared to those in the naïve secretome. CONCLUSION: We could, thus, conclude that the secretome released from miR-122-transfected ASCs has higher antifibrotic and anti-inflammatory properties than the naïve secretome. Because miR-122 transfection into ASCs provides a specific way of potentiating the antifibrotic properties of ASC secretome, it could be considered as an enhanced method for reinforcing secretome effectiveness.

Isolation of Secretome with Enhanced Antifibrotic Properties from miR-214-Transfected Adipose-Derived Stem Cells.

BACKGROUND: Secretome refers to the total set of molecules secreted or surface-shed by stem cells. The limitations of stem cell research have led numerous investigators to turn their attention to the use of secretome instead of stem cells. In this study, we intended to reinforce antifibrotic properties of the secretome released from adipose-derived stem cells (ASCs) transfected with miR-214. METHODS: We generated miR-214-transfected ASCs, and extracted the secretome (miR214-secretome) from conditioned media of the transfected ASCs through a series of ultrafiltrations. Subsequently, we intravenously injected the miR-214-secretome into mice with liver fibrosis, and determined the effects of miR-214-secretome on liver fibrosis. RESULTS: Compared with that by naïve secretome, liver fibrosis was ameliorated by intravenous infusion of miR-214-secretome into mice with liver fibrosis, which was demonstrated by significantly lower expression of fibrosis-related markers (alpha-smooth muscle actin, transforming growth factor-ß, and metalloproteinases-2) in the livers as well as lower fibrotic scores in the special stained livers compared with naïve secretome. The infusion of miR-214-secretome also led to lesser local and systemic inflammation, higher expression of an antioxidant enzyme (superoxide dismutase), and higher liver proliferative and synthetic function. CONCLUSION: MicroRNA-214 transfection stimulates ASCs to release the secretome with higher antifibrotic and anti-inflammatory properties. miR-214-secretome is thus expected to be one of the prominent ways of overcoming liver fibrosis, if further studies consistently validate its safety and efficiency.