Reprogramming of cancer stem cells into non-tumorigenic cells using stem cell exosomes for cancer therapy.

Cancer stem cells (CSCs) are a small population of cells with stem cell-like properties found in tumors. CSCs are closely associated with tumor heterogeneity, which influences tumor progress, metastasis, and drug resistance. Here, we propose a concept to enhance efficacy of cancer therapy through CSC reprogramming into non-tumorigenic cells using stem cell-derived exosomes with osteoinductive potential. We hypothesized that exosomes derived from osteogenic differentiating human adipose-derived stem cells (OD-EXOs) contain specific cargos capable of inducing osteogenic differentiation of CSCs. Quantitative RT-PCR analysis revealed that OD-EXOs enhanced the expression of osteogenic-related genes, such as alkaline phosphatase (ALPL), osteocalcin (BGLAP), and runt-related transcription factor 2 (RUNX2). In addition, expression of drug-resistance genes such as ATP binding cassette (ABC) transporter, the breast cancer gene family (BCRA1 and BCRA2), and the ErbB gene family were significantly decreased in OD-EXO-treated CSCs. Our findings suggest that OD-EXOs function as a biochemical cue for CSC reprogramming and contribute to overcoming therapeutic resistance.

Extracellular vesicles from adipose tissue-derived stem cells alleviate osteoporosis through osteoprotegerin and miR-21-5p.

Osteoporosis is one of the most common skeletal disorders caused by the imbalance between bone formation and resorption, resulting in quantitative loss of bone tissue. Since stem cell-derived extracellular vesicles (EVs) are growing attention as novel cell-free therapeutics that have advantages over parental stem cells, the therapeutic effects of EVs from adipose tissue-derived stem cells (ASC-EVs) on osteoporosis pathogenesis were investigated. ASC-EVs were isolated by a multi-filtration system based on the tangential flow filtration (TFF) system and characterized using transmission electron microscopy, dynamic light scattering, zeta potential, flow cytometry, cytokine arrays, and enzyme-linked immunosorbent assay. EVs are rich in growth factors and cytokines related to bone metabolism and mesenchymal stem cell (MSC) migration. In particular, osteoprotegerin (OPG), a natural inhibitor of receptor activator of nuclear factor-κB ligand (RANKL), was highly enriched in ASC-EVs. We found that the intravenous administration of ASC-EVs attenuated bone loss in osteoporosis mice. Also, ASC-EVs significantly inhibited osteoclast differentiation of macrophages and promoted the migration of bone marrow-derived MSCs (BM-MSCs). However, OPG-depleted ASC-EVs did not show anti-osteoclastogenesis effects, demonstrating that OPG is critical for the therapeutic effects of ASC-EVs. Additionally, small RNA sequencing data were analysed to identify miRNA candidates related to anti-osteoporosis effects. miR-21-5p in ASC-EVs inhibited osteoclast differentiation through Acvr2a down-regulation. Also, let-7b-5p in ASC-EVs significantly reduced the expression of genes related to osteoclastogenesis. Finally, ASC-EVs reached the bone tissue after they were injected intravenously, and they remained longer. OPG, miR-21-5p, and let-7b-5p in ASC-EVs inhibit osteoclast differentiation and reduce gene expression related to bone resorption, suggesting that ASC-EVs are highly promising as cell-free therapeutic agents for osteoporosis treatment.

Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes.

Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based therapy for regenerative medicine. In this study, stem cell EVs derived during differentiation are developed to use as cell-free therapeutic systems by inducing tissue-specific differentiation. EVs are isolated from human adipose-derived stem cells (HASCs) during white and beige adipogenic differentiation (D-EV and BD-EV, respectively) via tangential flow filtration. D-EV and BD-EV can successfully differentiate HASCs into white and beige adipocytes, respectively. D-EV are transplanted with collagen/methylcellulose hydrogels on the backs of BALB/c mice, and they produce numerous lipid droplets in injected sites. Treatments of BD-EV attenuate diet-induced obesity through browning of adipose tissue in mice. Furthermore, high-fat diet-induced hepatic steatosis and glucose tolerance are improved by BD-EV treatment. miRNAs are responsible for the observed effects of BD-EV. These results reveal that secreted EVs during stem cell differentiation into white adipocytes or beige adipocytes can promote cell reprogramming.

Small extracellular vesicles from human adipose-derived stem cells attenuate cartilage degeneration.

Osteoarthritis (OA) is a chronic degenerative disease of articular cartilage that is the most common joint disease worldwide. Mesenchymal stem cells (MSCs) have been the most extensively explored for the treatment of OA. Recently, it has been demonstrated that MSC-derived extracellular vesicles (EVs) may contribute to the potential mechanisms of MSC-based therapies. In this study, we investigated the therapeutic potential of human adipose-derived stem cells EVs (hASC-EVs) in alleviating OA, along with the mechanism. EVs were isolated from the culture supernatants of hASCs by a multi-filtration system based on the tangential flow filtration (TFF) system. The isolated EVs were characterised using dynamic light scattering (DLS), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and flow cytometry analysis. The hASC-EVs not only promoted the proliferation and migration of human OA chondrocytes, but also maintained the chondrocyte matrix by increasing type Ⅱ collagen synthesis and decreasing MMP-1, MMP-3, MMP-13 and ADAMTS-5 expression in the presence of IL-1ß in vitro. Intra-articular injection of hASC-EVs significantly attenuated OA progression and protected cartilage from degeneration in both the monosodium iodoacetate (MIA) rat and the surgical destabilisation of the medial meniscus (DMM) mouse models. In addition, administration of hASC-EVs inhibited the infiltration of M1 macrophages into the synovium. Overall results suggest that the hASC-EVs should be considered as a potential therapeutic approach in the treatment of OA.

Anti-gastric cancer effects of celecoxib, a selective COX-2 inhibitor, through inhibition of Akt signaling.

BACKGROUND AND AIM: Previously, we showed that treatment with celecoxib significantly reduced the number of viable gastric cancer cells, in a dose- and time-dependent manner. However, the specific anti-cancer effects of celecoxib on gastric cancer cells have not been clarified. The present in vitro study was carried out to investigate the mechanism involved in the anti-gastric cancer effects of celecoxib. METHODS: 3-(4,5-Dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was carried out after treating AGS cells (human gastric cancer cell line, ATCC CRL 1739) with celecoxib or indomethacin, and the effect of prostaglandin E(2) or LY294002 (PI3K inhibitor) was evaluated. Western blot analysis of tAkt (total Akt), pAkt (phosphorylated Akt), pGSK3beta (phosphorylated glycogen synthase kinase-3beta), pFKHR (phosphorylated forkhead transcriptional factor), and caspase-9 was carried out at various concentrations (0, 5, 10, 25, or 50 micromol/L) of celecoxib or indomethacin-treatment for 24 or 48 h in AGS cells. RESULTS: Celecoxib- or LY294002-induced cell death was found to occur in a dose-dependent manner in AGS cells, and these decreases were slightly recovered by the addition of PGE(2) (25 or 50 micromol/L). The expression of pAkt but not tAkt was lower in the celecoxib treated-AGS cells and the response was dose dependent (P < 0.05). The expression of pGSK3beta and pFKHR was also significantly decreased in the celecoxib treated-AGS cells. Procaspase 9 (47 kDa) was frequently cleaved into 37, 35 and 17 kDa fragments in the celecoxib-treatment group. However, these changes in cell signal transduction were not observed in the indomethacin treated-AGS cells. CONCLUSION: The anti-cancer effects of celecoxib on gastric cancer cells might be partly mediated by downregulation of Akt, GSK3beta, FKHR, and upregulation of caspase-9, in the mitochondrial apoptotic pathway.

Clinical significance of minimal change lesions of the esophagus in a healthy Korean population: a nationwide multi-center prospective study.

BACKGROUND AND AIM: At least half of the patients with typical reflux symptoms have non-erosive reflux disease (NERD). Minimal change lesions are commonly seen in the screening endoscopic examinations for individuals without clinically significant symptoms. We evaluated the correlation between minimal changes and symptoms in individuals visiting the hospital for routine health check-up by a nationwide survey in 2006. METHODS: Upper gastrointestinal endoscopic examinations as a health check-up were performed for 25,536 patients. Among them, symptom questionnaires were given in 23,350 patients without mucosal break or Barrett's esophagus. Endoscopic findings of the lower esophagus were divided into normal or minimal changes. Minimal changes in the present study included white turbid discoloration and Z-line blurring. RESULTS: Among a total of 25,536 subjects, reflux esophagitis was found in 2019 subjects (7.91%) and 3043 patients (11.9%) were classified as having minimal changes. History of gastroesophageal reflux disease (GERD) was more commonly found in individuals with minimal changes. Among the reflux-related symptoms, heartburn, acid regurgitation, globus sensation, and epigastric soreness were related to the minimal changes of the esophagus. Especially, individuals with globus sensation or epigastric soreness were more likely to have minimal changes compared to individuals without respective symptoms. Male gender, current smoker, history of H. pylori eradication, frequent stooping at work, hiatal hernia, and atrophic/metaplastic gastritis were found to be risk factors for minimal changes. CONCLUSION: The minimal changes were closely related with upper gastrointestinal symptoms and had similar risk factors for GERD, suggesting that minimal changes could be considered as early endoscopic findings of GERD.

Submucosal saline-epinephrine injection in colon polypectomy: appropriate indication.

BACKGROUND/AIMS: Opinions vary among endoscopists concerning the indications for submucosal saline-epinephrine injection for the prevention of postpolypectomy complications after colonic snare polypectomy. This study was performed to determine the appropriate indications for submucosal saline-epinephrine injection. METHODOLOGY: Clinical characteristics and complications (hemorrhage and perforation) in polypectomies were retrospectively evaluated. Postpolypectomy complications were analyzed in terms of demographic characteristics (age, gender), polyp characteristics (size, configuration, location, and histopathology), and the administration of submucosal saline-epinephrine injection. RESULTS: Total 1039 polypectomies were performed in 563 patients (age 59.8 +/- 10.1 years), and submucosal saline-epinephrine injection was performed in 679 polypectomies. Twenty seven episodes (2.6%) of hemorrhage and 3 cases (0.2%) of perforation occurred. Malignant adenoma, a rectal polyp, and procedure without submucosal saline-epinephrine injection increased the risk of hemorrhage with odds ratios of 10.48, 4.71, and 3.44, respectively. Furthermore, submucosal saline-epinephrine injection significantly reduced the risk of hemorrhage in sessile polyps and those > 8 mm in size and with odds ratio of 16.41 regardless of location or histopathology. The occurrence of postpolypectomy perforation was not associated with any clinical characteristics and method. CONCLUSIONS: Submucosal saline-epinephrine injection should be performed for sessile polyps and those > 8 mm in size, and might be performed optionally in other cases to prevent postpolypectomy hemorrhage.

[Clinical usefulness of fecal occult blood test as a screening method for asymptomatic patients with colon polyps].

BACKGROUND/AIMS: Detection of asymptomatic benign colon polyp is increasing because colonoscopy is widely used as a screening and diagnostic method. Fecal occult blood test is usually performed for the selection of patients requiring colonoscopy as well as mass screening for colon cancer. The aim of this study was to investigate the usefulness of fecal occult blood test performed prior to colonoscopy as a screening method of benign colon polyps. METHODS: Clinical characteristics of patients with polyps were evaluated according to the fecal occult blood test results in patients who underwent one-day fecal occult blood test and colonoscopic polypectomies from May 2003 to October 2004, retrospectively. RESULTS: A total of 942 colonoscopic polypectomies in 288 patients were evaluated. Fecal occult blood tests were positive only in 32 patients (11.1%). In univariate analysis, there was a significant difference in polyp size (p=0.02) and location (p=0.03) according to the presence of positive fecal occult blood tests. In addition, age of the patient (p=0.046), polyp size (mean, p=0.04; largest, p<0.01) and the number of polyps (p=0.045) were significantly different. However, in multivariate analysis, only polyp size larger than 20 mm was significantly related with positive fecal occult blood test with estimated odds ratio of 4.71. CONCLUSIONS: Fecal occult blood test has limitations as a screening test in asymptomatic patients with colon polyps, except for colon polyps larger than 20 mm in size.

Exosomes from differentiating human skeletal muscle cells trigger myogenesis of stem cells and provide biochemical cues for skeletal muscle regeneration.

Exosomes released from skeletal muscle cells play important roles in myogenesis and muscle development via the transfer of specific signal molecules. In this study, we investigated whether exosomes secreted during myotube differentiation from human skeletal myoblasts (HSkM) could induce a cellular response from human adipose-derived stem cells (HASCs) and enhance muscle regeneration in a muscle laceration mouse model. The exosomes contained various signal molecules including myogenic growth factors related to muscle development, such as insulin-like growth factors (IGFs), hepatocyte growth factor (HGF), fibroblast growth factor-2 (FGF2), and platelet-derived growth factor-AA (PDGF-AA). Interestingly, exosome-treated HASCs fused with neighboring cells at early time points and exhibited a myotube-like phenotype with increased expression of myogenic proteins (myosin heavy chain and desmin). On day 21, mRNAs of terminal myogenic genes were also up-regulated in exosome-treated HASCs. Moreover, in vivo studies demonstrated that exosomes from differentiating HSkM reduced the fibrotic area and increased the number of regenerated myofibers in the injury site, resulting in significant improvement of skeletal muscle regeneration. Our findings suggest that exosomes act as a biochemical cue directing stem cell differentiation and provide a cell-free therapeutic approach for muscle regeneration.

Clinical Analysis for Brain Tumor-Related Epilepsy during Chemotherapy for Systemic Cancer with Single Brain Metastasis.

PURPOSE: The purpose of this prospective observational study was to determine the incidence, patterns, and predisposing factors for brain tumor-related epilepsy (BTRE) during chemotherapy for systemic cancer with single brain metastasis (BM). MATERIALS AND METHODS: Between February 2006 and June 2010, 103 patients who underwent chemotherapy for systemic cancer with single BM were enrolled. We compared the clinical factors of patients and BM between patients with and without BTRE. We determined the number of patients with BTRE attacks, and seizure-free survival according to the following comparative groups: presence vs. absence of a history of BTRE; high-risk vs. low-risk groups; and presence vs. absence of disease-progression of BM. RESULTS: Ninety-three of 103 patients (90.3%) remained seizure-free during chemotherapy. The seizure-free rates were 88.9% and 91.0% among patients with or without a history of BTRE, respectively (p=0.694), 87.8% and 92.6% among high- and low-risk patients (p=0.427), respectively, and 62.5% and 98.7% among patients with or without disease-progression of BM (p=0.001), retrospectively. Based on multivariate analysis, the significance of abnormal findings on electroencephalogram (EEG) (p=0.017), and the absence of disease-progression of BM (p=0.001) had an association with seizure-free survival. CONCLUSION: The significance of abnormal findings on EEG, and disease-progression of BM play important roles in the development of BTRE during chemotherapy for systemic cancer with BM.

Prognostic factors of secondary cytoreductive surgery for patients with recurrent epithelial ovarian cancer.

OBJECTIVE: The objective of this study was to identify the prognostic factors of secondary cytoreductive surgery on survival in patients with recurrent epithelial ovarian cancer. METHODS: The medical records of all patients who underwent secondary cytoreductive surgery between May 2001 and October 2007 at the National Cancer Center, Korea were reviewed. Univariate and multivariate analyses were executed to evaluate the potential variables for overall survival. RESULTS: In total, 54 patients met the inclusion criteria. Optimal cytoreduction to <0.5 cm residual disease was achieved in 87% of patients who had received secondary cytoreductive surgery. Univariate analysis revealed that site of recurrence (median survival, 53 months for the largest tumors in the pelvis vs. 24 months for the largest tumors except for the pelvis; p=0.007), progression free survival (PFS) (median survival, 43 months for PFS>/=12 months vs. 24 months for PFS<12 months; p=0.036), and number of recurrence sites (median survival, 49 months for single recurred tumor vs 29 months for multiple recurred tumors; p=0.036) were significantly associated with overall survival. On multivariate analysis, prognostic factors that correlated with improved survival were site of recurrence (p=0.013), and PFS (p=0.043). CONCLUSION: In the author's analysis, a significant survival benefit was identified for the recurred largest tumors within the pelvis and PFS>/=12 months. Secondary cytoreductive surgery should be offered in selected patients and large prospective studies are needed to define the selection criteria for secondary cytoreductive surgery.