Stem Cell-Based or Cell-Free Gene Therapy in Chondrocyte Regeneration: Synovial Fluid-Derived Mesenchymal Stem Cell Exosomes.

BACKGROUND: Cartilage injuries are currently the most prevalent joint disease. Previous studies have emphasized the use of stem cells as the effective treatment for regenerating cartilage damage. OBJECTIVE: In this study, considering the difficulties of the cellular therapy method, it was hypothesized that human synovial fluid-derived mesenchymal stem cell (hSFMSC) exosomes as a SC source could be used to treat these injuries as a safer and cell-free therapeutic alternative procedure due to its direct relevance to cartilage regeneration. Moreover, this study aimed to determine the miRNA and target genes required for the formation of SC treatment combined with gene therapy in order to reveal the mechanism of cartilage regeneration and increase its effectiveness. METHODS: MSCs were characterized by flow cytometry, and immunocytochemical and differentiation analyses were done. To characterize functionally isolated exosomes, in vitro uptake analysis was performed. RT-qPCR was used to examine in terms of the advantages of cellular and cell-free therapy, mature human chondroblasts derived by differentiation from hSF-MSCs and human chondrocyte profiles were compared in order to demonstrate the above profile of hSF-MSCs and exosomes isolated from them, and the effectiveness of SC therapy in repairing cartilage damage. RESULTS: According to our findings, the expression level of hsa-miR-155-5p was found to be considerably higher in chondrocytes differentiated from human synovial fluid MSCs than in mature human chondrocytes. These findings were also supported by the TGF-signalling pathway and chondrogenesis marker genes. CONCLUSION: It was concluded that hSF-MSCs and exosomes can be used in the treatment of cartilage damage, and hsa-miR-155-5p can be used as a target miRNA in a new gene therapy approach because it increases the therapeutic effect on cartilage damage.

A multifunctional sateen woven dressings for treatment of skin injuries.

Cutaneous wounds with impaired healing such as diabetic ulcers and burns constitute major and rapidly growing threat to healthcare systems worldwide. Accelerating wound healing requires the delivery of biological factors that induce angiogenesis, support cellular proliferation, and modulate inflammation while minimizing infection. In this study, we engineered a dressing made by weaving of composite fibers (CFs) carrying mesenchymal stem cells (MSCs) and a model antibiotic using a scalable sateen textile technique. In this regard, two different sets of CFs carrying MSCs or an antimicrobial agent were used to generate a multifunctional dressing. According to cell viability and metabolic activity as CCK-8 and live/dead with qRT-PCR results, more than %90 the encapsulated MSCs remain viable for 28 days and their expression levels of the wound repair factors including ECM remodeling, angiogenesis and immunomodulatory maintained in MSCs post dressing manufacturing for 14 days. Post 10 days culture of the dressing, MSCs within CFs had 10-fold higher collagen synthesis (p < 0.0001) determined by hydroxyproline assay which indicates the enhanced healing properties. According to in vitro antimicrobial activity results determined by disk diffusion and broth microdilution tests, the first day and the total amount of release gentamicin loaded dressing samples during the 28 days were higher than determined minimal inhibition concentration (MIC) values for S. aureus and K. pneumonia without negatively impacting the viability and functionality of encapsulated MSCs within the dressing. The dressing is also flexible and can conform to skin curvatures making the dressing suitable for the treatment of different skin injuries such as burns and diabetic ulcers.

The potential therapeutic effect of klotho on cell viability in human colorectal adenocarcinoma HT-29 cells.

Klotho is an anti-aging, anti-inflammator, and anti-oxidative protein and has been shown to important role in tumorigenesis, proliferation, survival, autophagy, and resistance to tumor suppressor effects in several types of human cancers. In this study, we aimed to investigate possible anti-tümör and apoptotic effects of exogen klotho in human colorectal adenocarcinoma cells (HT-29) and healthy colon cells (CCD 841 CoN). The WST-8 test was used to determine the half-maximum inhibitory concentration (IC50) of the klotho protein. AO-PI fluorescent staining techniques and Annexin V-PI flow cytometry was utilized to observe and detect the apoptosis of cancer cells induced by klotho. Our results demonstrated that klotho had a cytotoxic effect against colorectal adenocarcinoma cells in a dose-dependent manner. Our Annexin V-PI flow cytometric and AO-PI fluorescent analyses showed that klotho induced quantitative and morphological changes that indicate apoptotic induction in the human colorectal adenocarcinoma. This study results proved for the first time that klotho may be an effective potential therapeutic agent that may be used in adjuvant therapy in human colorectal adenocarcinoma it does not affect selectively healthy colon cells and but leading cancer cells to apoptosis.

Bio-engineering of 3-D cell sheets for diabetic rats: Interaction between mesenchymal stem cells and beta cells in functional islet regeneration system.

Type 1 diabetes is an autoimmune disease that emerges with the destruction of beta cells of pancreatic Langerhans islets. Three different therapeutical approaches have been developed so far; pancreas transplantation, islet transplantation, and cell-based therapies. Bioengineering cell sheets for tissue generating is one of the latest approaches that have been used to construct cell-sheets instead of single cells so that it mimics the in vivo environments more. In this study, extra-hepatic functional islet tissue was constructed by transferring the 3-D beta cells and GFP labelled MSCs MSC sheets to the subcutaneous site of rats with STZ-induced diabetes. rBM-MSCs and beta cells were cultured on the 6-well PIPAAm culture dishes. Obtained rBM-MSCs as two-cell sheets and beta cells cultured in droplets with matrigel has transplanted into the dorsal subcutaneous area of diabetic rats. Fasting blood glucose levels and body weights were evaluated for 30 days after transplantation. Immunocytochemistry analysis for the anti-apoptotic, anti-inflammatory, and angiogenetic effects of MSCs on the 30th day of subcutaneous cell transplantation. All recipient rats transplanted with beta-cells with MSCs returned toward normoglycemia by day 5 and remained at this level for 30 days. Immunocytochemical analyses supported that the MSCs and beta cells preserved their viability and function. MSCs secrete cytokines and growth factors TGF-ß and IL-6; MSCs of the important features of the anti-apoptotic and anti-inflammatory properties, thanks to apoptosis of beta cells preserve graft explained by inhibition. In transplantation of MSCs induced angiogenesis and neovascularization, PECAM-1 and GFP positive simultaneously determining endothelial cells was observed indicating. Subcutaneous 3D beta-cell transplantation would be possible with the MSC-sheets as a feeder layer of beta cells. The beta-cell line is glucose-sensitive and has a high insulin release potential, and can be used as an alternative to islets in in vivo transplant studies.

Enhancement of Apo2L/TRAIL signaling pathway receptors by the activation of Klotho gene with CRISPR/Cas9 in Caco-2 colon cancer cells.

Human Klotho gene has many known functions such as anti-aging and anti-tumor, and decreased expression of this gene causes malignant formations in most types of cancer, including colon cancer. Interacting with TRAIL death receptors (DR4 and DR5) induces an apoptotic effect in cancer treatments by reducing the proliferation of cancer cells. The present study aimed to investigate downstream effect of overexpression of Klotho gene, which is known to have an antitumor effect on resistant human colon cancer cells, by examining its action on TRAIL death and decoy (DcR1 and DcR2) receptors for the first time. For this purpose, upregulation of human Klotho gene was achieved with CRISPR/Cas9-mediated system in resistant human colon cancer Caco-2 cells. To determine the effect of upregulation of Klotho gene on cancer cells evaluations with flow cytometry, WST-8, qRT-PCR, ELISA, and immunohistochemical analysis were performed. Then, Klotho gene was knocked out and its apoptotic effect was tested to find out whether it is due to overexpression of Klotho gene or not. Our results indicate that overexpression of Klotho gene in Caco-2 cells via CRISPR/Cas9-sensitized TRAIL death receptor DR4 suppresses the proliferation of cells by leading to apoptosis. Thus, this study conducted on apoptosis-resistant colon cancer cells may bring new insights about the role of Klotho gene in colon cancer.

The role of prokineticins in recurrent implantation failure.

The aim of the present study was to investigate the expression patterns of prokineticins (PROK) and prokineticin receptors (PROKR) in the endometrium of women with recurrent implantation failure (RIF). Fifteen (15) women with RIF and 15 fertile controls were enrolled in this study. Endometrial samples were taken from study participants with an endometrial biopsy cannula during the implantation window. Real time polymerase chain reaction and immunohistochemistry were used to determine PROK/PROKR mRNA expression and protein localization, respectively. PROK1 mRNA levels were 6.09 times higher compared to endometrial samples obtained from women with RIF than in samples obtained from fertile controls, whereas PROKR1 mRNA levels were 2.46 times lower in endometrial samples obtained from women with RIF than in samples from fertile controls. In addition, decreased PROKR1 was supported by immunohistochemistry analysis at protein level. There was no statistically significant difference between women with RIF and fertile controls regarding PROK2 and PROKR2 levels. Altered expression of the PROK1/PROKR1 system could be one of the numerous abnormalities in the endometrium of women with RIF.

The investigation of immunomodulatory effects of adipose tissue mesenchymal stem cell educated macrophages on the CD4 T cells.

Mesenchymal stem cells (MSCs) are strong immunomodulatory cells investigated in numerous clinical studies on fatal pathologies, such as graft versus host disease and autoimmune diseases; e.g., systemic lupus erythematosus, Crohn's disease, and ulcerative colitis. Macrophages are one of the critical cells linking the innate and adaptive immune system, and it has been shown that MSCs can differentiate between pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype of macrophages. However, it has not yet been fully clarified whether these differentiated macrophages are functional. In this study, we compared the immunomodulatory effects on the CD4 T cells of M1, M2a and M2c macrophages with the macrophages that directly and indirectly cultured with MSCs. We analyzed the changes in CD14, CD64, CD80, CD163 and CD200R expression to evaluate macrophage phenotypes, and the changes in CD4, IFN-g, IL-4, IL-17a and FoxP3 expression to evaluate T helper subsets using the FACS method. The changes in IL-1b, IL-4, IL-10, IL-12p70, IL-17a and IFN-g in the media supernatants were analyzed using the Luminex method. We also performed WST-1 and Caspase-3 ELISA analyses to observe the proliferation and apoptosis status of the T cells. MSCs were found to differentiate macrophages into a distinctive phenotype, which was close to the M2c phenotype, but was not considered as an M2c cell due to the low expression of CD163, a characteristic marker for M2c. While MEM-D, MEM-ID and MSCs showed similar inhibitory effects on the Th2 and Th17 cells, the most significant increase in Treg cell frequencies was seen in MEM-D cells. Macrophages can alter their phenotypes and functions according to the stimuli from the environment. The fact that macrophages educated with MSCs suppressed the production of all the cytokines we evaluated even after the removal of MSCs suggests that these cells may be differentiated by MSCs into a suppressive macrophage subgroup. However, the Treg cell activation caused by direct interactions between MSCs and macrophage cells may be the most prominent observation of this study compared to previous work. As a result, according to our data, the interactions between MSCs and macrophages may lead to differentiation of macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress the T lymphocyte subgroups at least as effectively as MSCs. However, our data obtained from in vitro experiments should be supported by future in vivo studies.

The paracrine immunomodulatory interactions between the human dental pulp derived mesenchymal stem cells and CD4 T cell subsets.

Mesenchymal stem cells (MSCs) have strong immunomodulatory properties, however these properties may show some differences according to the tissue type of their isolate. In this study we investigated the paracrine interactions between human DP derived MSCs (hDP-MSCs) and the CD4+ T helper cell subsets to establish their immunomodulatory mechanisms. We found that the CD4+-Tbet+ (Th1) and CD4+-Gata3+ (Th2) cells were suppressed by the hDP-MSCs, but the CD4+-Stat3+ (Th17) and CD4+-CD25+-FoxP3+ (Treg) cells were stimulated. The expressions of T cell specific cytokines interferon gamma (IFN-g), interleukin (IL)-4 and IL-17a decreased, but IL-10 and transforming growth factor beta-1 (TGF-b1) increased with the hDP-MSCs. The expressions of indoleamine-pyrrole 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), soluble human leukocyte antigen G (sHLA-G) derived from hDP-MSCs slightly increased, but hepatocyte growth factor (HGF) significantly increased in the co-culture groups. According to our findings, the hDP-MSCs can suppress the Th1 and Th2 subsets but stimulate the Th17 and Treg subsets. The Stat3 expression of Th17 cells may have been stimulated by the HGF, and thus the pro-inflammatory Th17 cells may have altered into the immunosuppressive regulatory Th17 cells. Further prospective studies are needed to confirm our findings.

Immunoregulatory effects of human dental pulp-derived stem cells on T cells: comparison of transwell co-culture and mixed lymphocyte reaction systems.

BACKGROUND AIMS. Studies performed using human and animal models have indicated the immunoregulatory capability of mesenchymal stromal cells in several lineages. We investigated whether human dental pulp-derived stem cells (hDP-SC) have regulatory effects on phytohemagglutinin (PHA)-activated CD3(+) T cells. We aimed to define the regulatory mechanisms associated with hDP-SC that occur in mixed lymphocyte reaction (MLR) and transwell systems with PHA-CD3(+) T cells and hDP-SC at a ratio of 1:1. METHODS. Proliferation, apoptosis and pro- and anti-inflammatory cytokines of PHA-CD3(+)T cells, the expression of Regulatory T cells (Treg) markers and some regulatory factors related to hDP-SC, were studied in Both transwell and MLR are co-cultures systems. RESULTS. Anti-proliferative and apoptotic effects of hDP-SC were determined in co-culture systems. Elevated expression levels of human leukocyte antigen (HLA)-G, hepatocyte growth factor (HGF)-ß1, intracellular adhesion molecule (ICAM-1)-1, interleukin (IL)-6, IL-10, transforming growth factor (TGF)-ß1, vascular adhesion molecule (VCAM)-1 and vascular endothelial growth factor (VEGF) by hDP-SC were detected in the co-culture systems. We observed decreased expression levels of pro-inflammatory cytokines [interferon (IFN)-γ, IL-2, IL-6 receptor (R), IL-12, Interleukin-17A (IL-17A), tumor necrosis factor (TNF)-α] and increased expression levels of anti-inflammatory cytokine [inducible protein (IP)-10] from PHA-CD3(+) T cells in the transwell system. Expression of Treg (CD4(+) CD25(+) Foxp3(+)) markers was significantly induced by hDP-SC in both co-culture systems. We observed apoptosis of PHA-CD3(+) T cells with 24 h using time-lapse camera photographs and active caspase labeling; it is likely that paracrine soluble factors and molecular signals secreted by hDP-SC led this apoptosis. CONCLUSIONS. We suggest that hDP-SC have potent immunoregulatory functions because of their soluble factors and cytokines via paracrine mechanisms associated with PHA-CD3(+) T cells, which could contribute to clinical therapies.

Isolation and in vitro characterisation of dental pulp stem cells from natal teeth.

Dental pulp stem cells were primarily derived from the pulp tissues of exfoliated deciduous teeth, primary incisors and permanent third molar teeth. The aim of this study was to isolate and extensively characterise SCs derived from human natal dental pulp (hNDP). For characterisation, proliferation capacity, phenotypic properties, ultrastructural and differentiation characteristics and gene expression profiles were utilised. A comparison was done between the properties of NDP-SCs and the properties of mesenchymal stem cells (MSCs) from bone marrow (BM) of the human. Stem cells isolated from hNDP and hBM were analysed by flow cytometry, reverse transcriptase-PCR, Real Time-PCR, and immunocytochemistry. Both cell lines were directionally differentiated towards adipogenic, osteogenic chondrogenic, myogenic and neurogenic lineages. hNDP-SCs and hBM-MSCs expressed CD13, CD44, CD90, CD146 and CD166, but not CD3, CD8, CD11b, CD14, CD15, CD19, CD33, CD34, CD45, CD117, and HLA-DR. Ultrastructural characteristics of hNDP-SCs showed more developed and metabolically active cells. hNDP-SCs and hBM-MSCs expressed some adipogenic (leptin, adipophilin and PPARgamma), myogenic (desmin, myogenin, myosinIIa, and alpha-SMA), neurogenic (gamma-enolase, MAP2a,b, c-fos, nestin, NF-H, NF-L, GFAP and betaIII tubulin), osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, and type I collagen) and chondrogenic (type II collagen, SOX9) markers without any stimulation towards differentiation under basal conditions. Embryonic stem cell markers Oct4, Rex-1, FoxD-3, Sox2, and Nanog were also identified. The differentiation potential of hNDP-SCs and hBM-MSCs to adipogenic, osteogenic, chondrogenic, myogenic and neurogenic was shown. This report described the first successful isolation and characterisation of hNDP-SCs.

Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and immunophenotypic markers.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can differentiate into many lineages. Although the growing interest in BM-MSCs has led to a number of characterization studies, some important biochemical and immunohistochemical properties are still lacking. In this study, morphological and immunophenotypic properties of BM-MSCs were examined in detail. Differentiation potential and growth kinetics of adult rat BM-MSCs were also determined. Immunohistochemistry and RT-PCR results indicated that BM-MSCs expressed myogenic (desmin, myogenin, myosin IIa, and alpha-SMA), neurogenic (gamma-enolase, MAP2a,b, c-fos, nestin, GFAP and beta III tubulin), and osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, BMP-2, BMP-4 and type I collagen) markers without stimulation towards differentiation. These expression patterns indicated why these cells can easily differentiate into multiple lineages both in vitro and in vivo. Ultrastructural characteristics of rBM-MSCs showed more developed and metabolically active cells.