Human Adipose Mesenchymal Stem Cell-Derived Exosomes: A Key Player in Wound Healing.

BACKGROUND: Human adipose-derived mesenchymal stem cells (AMSCs) are an attractive resource for wound healing because their regenerative capacity improves injury repair. Recently, stem cell-derived exosomes have been shown to play a positive role in stem cell-based therapies. However, the effects of exosomes derived from AMSCs (AEXOs) on wound healing are unclear. In this study, we aimed to examine the role of AEXOs in attenuating inflammation and explore their effects in normal wound healing. METHODS: We isolated exosomes from AMSCs and established a cellular model of inflammation by treatment with the inflammatory cytokines, interferon gamma and tumor necrosis factor alpha, to determine whether AEXOs can inhibit inflammation. We examined the wound healing effects of AEXOs in in vitro wound healing models and performed a miRNA array to understand the role of AEXOs in inflammation and wound healing. RESULTS: A significant difference was observed in wound closure and the expression of anti-inflammatory and wound-healing-related factors between control and AEXO-treated cells. CONCLUSION: Our results showed that besides alleviating the inflammation response, AEXOs also promote wound healing. Thus, AEXOs represent a novel, stem-cell-based, therapeutic strategy for wound healing.

Adipose-Derived Mesenchymal Stem Cells Promote M2 Macrophage Phenotype through Exosomes.

Accumulating evidence has shown that the paracrine factors derived from mesenchymal stem cells (MSCs) are capable of regulating the immune system via interaction with various immune cells. In this study, adipose-derived MSCs (AdMSCs) and human peripheral blood monocytes (PBMCs) were isolated and cultured to examine the effects of MSC-induced macrophages (iMΦ) on inflammation and immune modulation. Indirect coculture with MSCs increased the expression of arginase-1 and mannose receptor (CD206), markers of activated M2 macrophages, in the PBMCs demonstrating that MSC-secreted factors promoted M2-MΦ polarization. Additionally, iMΦ exhibited a similar higher inhibitory effect on the growth of activated T cells compared to that in the other groups (AdMSCs only, AdMSCs plus iMΦ), implying that iMΦ can play a sufficient functional role. Interestingly, the population of FoxP3 Treg cells significantly increased when cocultured with iMΦ, suggesting that iMΦ have an immunomodulatory effect on the Treg cells through the modulation of the FoxP3 expression. Notably, iMΦ expressed high levels of immunosuppressive and anti-inflammatory cytokines, namely IL-10 and TSG-6. Furthermore, we confirmed that the AdMSC-derived exosomes modulated macrophage polarization by upregulating the expression of M2 macrophage markers. Conclusively, our results suggest that iMΦ play a significant role in regulating the immunomodulatory- and inflammatory-mediated responses. Thus, iMΦ may be used as a novel stem cell-based cell-free therapy for the treatment of immune-mediated inflammatory disorders.

The cryptochrome inhibitor KS15 enhances E-box-mediated transcription by disrupting the feedback action of a circadian transcription-repressor complex.

AIMS: We have previously identified a chemical scaffold possessing 2-ethoxypropanoic acid (designated as KS15) that directly binds to the C-terminal region of cryptochromes (CRYs: CRY1 and CRY2) and enhances E-box-mediated transcription. However, it is still unclear how KS15 impairs the feedback actions of the CRYs and which chemical moieties are functionally important for its actions. MAIN METHODS: The E-box-mediated transcriptional activities were mainly used to examine the effects of KS15 and its derivatives. Co-immunoprecipitation assays accompanied by immunoblotting were employed to monitor protein-protein associations. We also examined the effects of KS15 and selected derivatives on circadian molecular rhythms in cultured cells. KEY FINDINGS: The present study shows that KS15 inhibits the interaction between CRYs and Brain-Muscle-Arnt-Like protein 1 (BMAL1), thereby impairing the feedback actions of CRYs on E-box-dependent transcription by CLOCK:BMAL1 heterodimer, an indispensable transcriptional regulator of the mammalian circadian clock. Subsequent structure-activity relationship analyses using a well-designed panel of derivatives identified the structural requirements for the effects of KS15 on CRY-evoked regulation of E-box-mediated transcription. We found that KS15 and several derivatives significantly reduce the amplitude and delayed the phase of molecular circadian rhythms in fibroblast cultures. SIGNIFICANCE: Taken together, our results provide valuable information on the molecular mode-of-action as well as the chemical components of the CRYs inhibitor that pharmacologically impact on the transcriptional activity of the CLOCK:BMAL1 heterodimer.

Poly-L-lysine Prevents Senescence and Augments Growth in Culturing Mesenchymal Stem Cells Ex Vivo.

Mesenchymal stem cells (MSCs) possess great therapeutic potential. Efficient in vitro expansion of MSCs is however necessary for their clinical application. The extracellular matrix (ECM) provides structural and biochemical support to the surrounding cells, and it has been used as a coating substrate for cell culture. In this study, we have aimed to improve the functionality and stemness of MSCs during culture using poly-L-lysine (PLL). Functionality of MSCs was analysed by cell cycle analysis, differentiation assay, ß-galactosidase staining, and RT-PCR. Furthermore, we assessed the global gene expression profile of MSCs on uncoated and PLL-coated plates. MSCs on PLL-coated plates exhibited a faster growth rate with increased S-phase and upregulated expression of the stemness markers. In addition, their osteogenic differentiation potential was increased, and genes involved in cell adhesion, FGF-2 signalling, cell cycle, stemness, cell differentiation, and cell proliferation were upregulated, compared to that of the MSCs cultured on uncoated plates. We also confirmed that MSCs on uncoated plates expressed higher ß-galactosidase than the MSCs on PLL-coated plates. We demonstrate that PLL provides favourable microenvironment for MSC culture by reversing the replicative senescence. This method will significantly contribute to effective preparation of MSCs for cellular therapy.

Comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow, umbilical cord blood, placenta and adipose tissue.

Mesenchymal stem cells (MSCs) are clinically useful due to their capacity for self-renewal, their immunomodulatory properties and tissue regenerative potential. These cells can be isolated from various tissues and exhibit different potential for clinical applications according to their origin, and thus comparative studies on MSCs from different tissues are essential. In this study, we investigated the immunophenotype, proliferative potential, multilineage differentiation and immunomodulatory capacity of MSCs derived from different tissue sources, namely bone marrow, adipose tissue, the placenta and umbilical cord blood. The gene expression profiles of stemness-related genes [octamer-binding transcription factor 4 (OCT4), sex determining region Y-box (SOX)2, MYC, Krüppel-like factor 4 (KLF4), NANOG, LIN28 and REX1] and lineage­related and differentiation stage-related genes [B4GALNT1 (GM2/GS2 synthase), inhibin, beta A (INHBA), distal-less homeobox 5 (DLX5), runt-related transcription factor 2 (RUNX2), proliferator­activated receptor gamma (PPARG), CCAAT/enhancer-binding protein alpha (C/EBPA), bone morphogenetic protein 7 (BMP7) and SOX9] were compared using RT-PCR. No significant differences in growth rate, colony-forming efficiency and immunophenotype were observed. Our results demonstrated that MSCs derived from bone marrow and adipose tissue shared not only in vitro tri-lineage differentiation potential, but also gene expression profiles. While there was considerable inter-donor variation in DLX5 expression between MSCs derived from different tissues, its expression appears to be associated with the osteogenic potential of MSCs. Bone marrow-derived MSCs (BM-MSCs) significantly inhibited allogeneic T cell proliferation possibly via the high levels of the immunosuppressive cytokines, IL10 and TGFB1. Although MSCs derived from different tissues and fibroblasts share many characteristics, some of the marker genes, such as B4GALNT1 and DLX5 may be useful for the characterization of MSCs derived from different tissue sources. Collectively, our results suggest that, based on their tri-lineage differentiation potential and immunomodulatory effects, BM-MSCs and adipose tissue-derived MSCs (A-MSCs) represent the optimal stem cell source for tissue engineering and regenerative medicine.

Noninvasive photodetachment of stem cells on tunable conductive polymer nano thin films: selective harvesting and preserved differentiation capacity.

Viable mesenchymal stem cells (MSCs) were efficiently and selectively harvested by near-infrared (NIR) light using the photothermal effect of a conductive polymer nano thin film. The poly(3,4-ethylenedioxy thiophene) (PEDOT)-coated cell culture surfaces were prepared via a simple and fast solution-casting polymerization (SCP) technique. The absorption of PEDOT thin films in the NIR region was effectively triggered cell harvesting upon exposure to an NIR source. By controlling the NIR absorption of the PEDOT film through electrochemical doping or growing PEDOT with different thin film thickness from 70 to 300 nm, the proliferation and harvesting of MSCs on the PEDOT surface were controlled quantitatively. This light-induced cell detachment method based on PEDOT films provides the temporal and spatial control of cell harvesting, as well as cell patterning. The harvested stem cells were found to be alive and well proliferated despite the use of temperature increase by NIR. More importantly, the harvested MSCs by this method preserved their intrinsic characteristics as well as multilineage differentiation capacities. This PEDOT surfaces could be used for repetitive culture and detachment of MSCs or for efficient selection or depletion of a specific subset from heterogeneous population during culture of various tissue-derived cells because there were no photodegradation and photobreakage in the PEDOT films by NIR exposure.

Sulforaphane-PLGA microspheres for the intra-articular treatment of osteoarthritis.

Sulforaphane (SFN) is a member of the isothiocyanate family that has anti-inflammatory action as well as anti-carcinogenic properties. The authors have devised an intra-articular injectable SFN-PLGA microsphere system that can be used for treating osteoarthritis (OA). The purpose of this study was to evaluate the in vitro and in vivo efficacy of the SFN-PLGA microsphere system. Articular chondrocytes were obtained from knee OA patients and were cultured in monolayers. The optimal concentration of SFN was obtained and the dose of SFN-PLGA microspheres was determined based on the concentration. The in vitro anti-inflammatory effect on markers such as cyclooxygenase (COX)-2, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5, and matrix metalloproteinase (MMP)-2 was assessed by real-time PCR and Western blotting. The in vivo therapeutic effect of SFN-PLGA microspheres was investigated using surgically-induced rat OA model. Treatment with SFN-PLGA microspheres inhibited the mRNA and protein expression of COX-2, ADAMTS-5 and MMP-2 induced by LPS in articular chondrocytes. Intraarticular SFN-PLGA microspheres delayed the progression of surgically-induced osteoarthritis in rats. In conclusion, SFN-PLGA microspheres can be a useful injectable delivery system for treating osteoarthritis.

Epigenetics in osteoarthritis and its implication for future therapeutics.

INTRODUCTION: Changes occurring in the chondrocyte gene that control articular cartilage are important for the onset and progression of osteoarthritis (OA). However, actual development of the disease may be also controlled by changes in epigenome. AREAS COVERED: Topics include the association of the three components of epigenetic modification, i.e., DNA methylation, histone modification, and microRNA expression, with the pathogenesis and progression of OA. The cross talk between genetics and epigenetics as well as the implication of epigenetics as a therapeutic measure for OA is also introduced. EXPERT OPINION: Epigenetic markers that detect various chondrocyte phenotypes, including those involving chondrogenic differentiation, articular cartilage homeostasis, and progression of OA, may provide a novel means to detect early OA. Recent report of dietary supplement such as glucosamine that prevents demethylation of promoters of inflammatory cytokine is encouraging. Although already available, the toxicity and off-target side effects of histone deacetylase inhibitors are concerns for benign nonlethal disease like OA. miRNA-based treatment may present another therapeutic modality without potentially detrimental off-target side effects. Future studies are needed to search for additional miRNA that can modulate the course of OA and to identify key targets of currently known miRNA that impact OA pathogenesis and disease progression.

Duration of in vitro storage affects the key stem cell features of human bone marrow-derived mesenchymal stromal cells for clinical transplantation.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have the ability to self-renew and differentiate into various cell types. Their plasticity and easy availability make them promising candidates for regenerative medicine. However, for successful clinical application, MSCs need to be expanded under a Good Manufacturing Practices-compliant system to obtain a large quantity of these cells. Although the viability and potency of these in vitro-expanded MSCs need to be maintained during preparation and transportation before transplantation, these characteristics have not thoroughly been examined. Our goal in this study was to standardize MSC preparation and storage before their clinical application to ensure reproducible quality and potency for their clinically intended purpose. METHODS: We examined the viability, self-renewal capacity and differentiation capability of MSCs on short-term in vitro storage in saline or dextrose solution at 4°C and room temperature. RESULTS: MSCs harvested and suspended in saline for 1-2 h showed >90% viability regardless of storage temperature. However, when cells were stored for >2 h in saline, their viability decreased gradually over time. The viability of cells in dextrose deteriorated rapidly. MSCs lost colony-forming unit and differentiation capacities rapidly as storage time increased. Collectively, we found that a storage period >2 h resulted in a significant decrease in cell viability, cell proliferation capacity and differentiation potency. CONCLUSIONS: Storage of culture-harvested MSCs for >2 h is likely to result in suboptimal MSC-mediated tissue regeneration because of decreased cell viability and differentiation capacity.

Comparison of endothelial progenitor cells in Parkinson's disease patients treated with levodopa and levodopa/COMT inhibitor.

BACKGROUND: Levodopa treatment in Parkinson's disease (PD) increases in serum homocysteine levels due to its metabolism via catechol O-methyltransferase. Endothelial progenitor cells (EPCs) have the capacity to differentiate into mature endothelial cells and are markers for endothelial functions and cardiovascular risks. Along with traditional vascular risk factors, hyperhomocysteinemia is known to decrease the level of EPCs. In the present study, we hypothesized that that levodopa-induced hyperhomocysteinemia leads to a change in EPC levels. METHODOLOGY/PRINCIPAL FINDINGS: We prospectively enrolled PD patients who had been prescribed either levodopa/carbidopa (PD-L group, n = 28) or levodopa/carbidopa/COMT inhibitor (PD-LC group, n = 25) for more than 1 year. The number of circulating EPCs was measured by flow cytometry using dual staining of anti-CD34 and anti-KDR antibodies. The EPCs were divided into tertiles based on their distributions and a logistic regression analysis was used to estimate independent predictors of the highest tertile of EPCs. The number of endothelial progenitor cells was significantly decreased in PD-L patients (118±99/mL) compared with either PD-LC patients (269±258/mL, p = 0.007) or controls (206±204/mL, p = 0.012). The level of homocysteine was significantly increased in PD-L patients (14.9±5.3 µmol/L) compared with either PD-LC patients (11.9±3.0 µmol/L, p = 0.028) or controls (11.1±2.5 µmol/L, p = 0.012). The level of homocysteine was negatively correlated with endothelial progenitor cell levels (r = -0.252, p = 0.028) and was an independent predictor of the highest tertile of endothelial progenitor cell levels (OR; 0.749 [95% CI: 0.584-0.961]). CONCLUSIONS/SIGNIFICANCE: These data indicate that a higher consumption of EPC for restoration of endothelial damage may be associated with chronic levodopa treatment in PD patients.

Monocyte enrichment from leukapheresis products by using the Elutra cell separator.

BACKGROUND: Dendritic cells (DCs), used in clinical trials for cancer immunotherapy, require processing on an expanded scale to conform to current good manufacturing practice guidelines. This study evaluated a large-scale monocyte enrichment procedure with a commercially available cell separator (Elutra, Gambro BCT) and analyzed the capacity of enriched monocytes to differentiate into DCs. STUDY DESIGN AND METHODS: Mononuclear cells were collected in two patients with malignant melanoma and seven healthy donors by leukapheresis. Continuous-counterflow elutriation with the Elutra was performed to enrich and purify monocytes from leukapheresis products. Purity and recovery of enriched monocytes were analyzed by flow cytometry. DCs were generated from the elutriated monocytes and characterized by phenotypic surface marker and stimulatory capacity in an allogeneic mixed lymphocyte reaction. RESULTS: In the leukapheresis products, the total MNC count was 7.3 x 10(9) +/- 0.7 x 10(9) and the mean percentage of CD14+ monocytes was 16.5 +/- 3.8 percent, which increased to 68.9 +/- 7.4 percent after elutriation with the Elutra. The mean monocyte recovery was 94.3 percent. Elutriated monocytes were successfully cultured into phenotypically and functionally mature DCs. CONCLUSION: These results indicate that the Elutra cell separator allows for fast and easy enrichment of monocytes within a closed system. Furthermore, these monocytes can be differentiated into functionally mature DCs. Compared to plastic adherence and immunomagnetic selection methods, the elutriation procedure is inexpensive, efficient, and very effective.

Analysis of gene expression profiles of hepatocellular carcinomas with regard to 18F-fluorodeoxyglucose uptake pattern on positron emission tomography.

PURPOSE: 18F-fluorodeoxyglucose (FDG) uptake on positron emission tomography (PET) scan has been found to reflect tumour aggressiveness and prognosis in various types of cancer. In this study, the gene expression profiles of hepatocellular carcinomas (HCCs) were evaluated to determine whether HCCs with high 18F-FDG uptake have more aggressive biological potential than those with low uptake. METHODS: Surgical specimens were obtained from ten patients with HCC (six males and four females, age range 38-68 years). The tumour samples were divided into two groups based on the 18F-FDG PET scan findings: high 18F-FDG uptake (n=4) and low 18F-FDG uptake (n=6). RESULTS: The pathological tumour grade was closely correlated with the 18F-FDG uptake pattern: HCCs with high 18F-FDG uptake were pathologically Edmondson-Steiner grade III, while those with low uptake were either grade II or grade II with a focal area of grade III. The total RNA was extracted from the frozen tissues of all HCCs (n=10) and adjacent non-cancerous tissue (n=7). The gene expression profiles were evaluated using an oligoDNA microarray. The HCCs with high 18F-FDG uptake showed increased expression of 11 genes--including vascular cell adhesion molecule-1, vinexin beta and core 1 UDP-galactose:N-acetylgalactosamine-alpha-R-beta 1,3-galactosyltransferase and the natural killer cell inhibitory receptor--compared to those with low uptake (p<0.005). Nine genes, including regulator of mitotic spindle assembly 1, grb2-related adaptor protein and beta-1,3-n-acetylglucosaminyltransferase, were repressed. CONCLUSION: Gene expression is closely related to cell survival, cell-to-cell adhesion or cell spreading; therefore, HCCs with high 18F-FDG uptake appear to have more aggressive biological properties than those with low uptake.