The effects of crocetin on oxidative stress induced ARPE-19 cells by H2O2.

The anti-apoptotic and antioxidant effects of crocetin was aimed to investigate on the oxidative damage model of ARPE-19 cells. The oxidative damage in ARPE cells was developed by H2O2 treatment at 800 µM. Different doses of crocetin (1-80 µM) were applied for 24 h, and the effects on viability were evaluated to find out the optimum drug dose. At first, three effective doses of crocetin (10, 20, 40 µM) on cell viability were selected for further analyses. The levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) were determined, and the expression of pro-apoptotic Bax gene and anti-apoptotic Bcl-2 gene were evaluated. The most effective crocetin dose on cell viability was found to be 10 µM. After the H2O2 treatment, SOD and GSH were decreased and MDA were increased significantly (p = 0.011, 0.037, 0.018, respectively). Following the crocetin treatment at 10 µM, SOD and GSH activities were improved compared to the no drug group; and MDA level was declined remarkably (p = 0.022, 0.019, 0.029, respectively). The Bcl-2 level was significantly decreased (p < 0.01), while the Bax1 and Nrf2 expression and ROS level was increased significantly in the damage model group (p < 0.01). After the drug treatment, the Bax1 and Nrf2 expression level were decreased in all groups (p < 0.01). The increase in Bcl-2 expression was significant in crocetin 40 µM (p < 0.05) and the decrease in ROS level were significant in 20 µM and 40 µM doses of crocetin (p < 0.05). It has been shown that crocetin might be used as an antioxidant and anti-apoptotic agent on the hindering the effect of the oxidative damage. Following the development of the oxidative stress in the cells, crocetin reversed the damage signals. By the in vitro tests, it was shown that crocetin might be considered as an effective molecule to be used in the AMD treatment.

Evaluation of clinical and histological effects of KGF-2 and NGF on corneal wound healing in an experimental alkali burn rabbit model.

Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-ß (TGF-ß) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-ß was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.

The therapeutical effects of damage-specific stress induced exosomes on the cisplatin nephrotoxicity IN VIVO.

Cisplatin is one of the metal containing drugs for the solid cancer treatments. However, its side-effects limit its application in the cancer treatment. Stem cell therapy is a promising treatment for the tissue damage caused by the chemotherapeutic agents, like cisplatin. Exosomes secreted by mesenchymal stem cells (MSCs) could be used for cell-free regenerative treatment, but their potency and reproducibility are questionable. In this study, the microenvironment of the renal tubular epithelial cells was mimicked by coculture of endothelial-, renal proximal tubule epithelial- and fibroblast cells. Cisplatin was applied to this tricell culture model, and the secreted rescue signals were collected and used to induce MSCs. From these stress-induced MSCs, the (stress-induced) exosomes were collected and used for the cell-free therapeutic treatment of cisplatin-treated rats with acute kidney injury. The composition of the stress-induces exosomes was compared with the non-induced exosomes and found that the expression of some critical factors for cell proliferation, repair mechanism and oxidative stress was improved. The cisplatin-damaged renal tissue showed substantial recovery after the treatment with stress-induced exosomes compared to the treatment with non-induced exosomes. Although, the non-induced exosomes showed their activity mostly as cytoprotective, the induced exosomes further involved actively in the tissue regeneration, like MSCs. It was shown that the exosomes could be reprogrammed to improve their therapeutic effect to be used in cell-free regenerative medicine. Further, cisplatin-induced tissue damage in the kidney might be effectively prevented and used for tissue regeneration by use of induced exosomes generated for a particular damage.

Protective effects of citicoline-containing eye drops against UVB-Induced corneal oxidative damage in a rat model.

It has been reported that citicoline increases antioxidant activity in some tissues. However, the effect of citicoline on corneal wound-healing has not yet been demonstrated. The aim was to investigate the protective effects of citicoline on ultraviolet B (UVB) radiation-induced corneal oxidative damage in a rat model. Four groups (eight animals each) were investigated: controls; UVB only; UVB/citicoline; and citicoline only. Corneal oxidative damage was induced by exposure to UVB radiation at 560 µW/cm2 for five days in the UVB-exposed groups and 1% citicoline eye drops were applied (3xday) for eight days in the two citicoline groups. Corneal surface damage was evaluated by opacity and fluorescein staining. Corneal injury was assessed biochemically by measuring the concentrations of glutathione (GSH) and malondialdehyde (MDA) and the activity of corneal superoxide dismutase (SOD) and catalase. Matrix metalloproteinase (MMP) -2 and -9 and caspase-3 were evaluated by immunofluorescent staining and microscopic examination and by Western blot analysis. Corneal gene expression analysis was performed for vascular endothelial growth factor (VEGF), interleukin-1 beta (IL-1ß) and transforming growth factor-beta (TGF-ß). UVB radiation caused significant epithelial damage and evident opacity in the cornea, together with a local decrease in SOD, catalase and GSH activity. Corneal MDA concentrations increased with UVB exposure. The UVB/Citicoline group had significantly less corneal damage, greater SOD, catalase and GSH activity, and decreased MDA concentrations compared to the UVB only group (p < 0.05). Expression of TGF-ß, IL-1ß and VEGF was significantly lower in the citicoline/UVB group compared to the UVB group (p < 0.05). Interestingly, TGF-ß expression was lower in the citicoline only group compared with controls. Immunfluorescent staining and Western blot analysis showed increased MMP-2, -9 and caspase-3 in the UVB only group compared with the UVB/citicoline group. It was shown that citicoline treatment may be effective in suppressing oxidative stress and controlling inflammation in UVB corneal injury.

Effects of different pulp-capping materials on cell death signaling pathways of lipoteichoic acid-stimulated human dental pulp stem cells.

PURPOSE: This study aimed to evaluate the response of dental pulp stem cells (DPSCs) cultured with and without lipoteichoic acid (LTA) to different pulp-capping materials. METHODS: The cells were cultured and seeded in 6-well plates and exposed to 1% LTA solution. Dycal, ProRoot MTA and Biodentine materials were applied on cells and all groups were evaluated by cell proliferation, viability, cell cycle and cell death signaling pathways for 24 and 72 h. RESULTS: LTA + Dycal treatment significantly inhibited the proliferation of DPSCs and increased the apoptosis rate of cells more than the other groups at 72 h. Compared to other groups, LTA + Dycal treatment significantly increased the levels of Caspase-3 and AKT and decreased the levels of p-AKT. CONCLUSIONS: The results of this study revealed that all tested materials caused apoptosis in DPSCs via an extrinsic apoptotic pathway. The DPSCs showed an early apoptosis response to the Dycal and a late apoptosis response to the ProRoot MTA and Biodentine treatments. LTA led autophagy and inhibited the proliferation of DPSCs. ProRoot MTA and Biodentin eliminated the LTA's bioactivity with higher efficiency than Dycal.

Effect of bone marrow and adipose tissue-derived mesenchymal stem cells on the natural course of corneal scarring after penetrating injury.

In the present study, we investigate and compare the efficacy of bone marrow- and adipose tissue-derived mesenchymal stem cell (MSCs) in corneal wound healing. A penetrating injury was created in the right corneas of Wistar rats (n = 40). Ten microliters of phosphate-buffered solution (PBS) containing 2 × 10(5) green fluorescent protein (GFP) labeled bone-marrow-derived MSCs to group 1 (n = 15), 10 µl of PBS containing 2 × 10(5) GFP-labeled adipose-tissue-derived MSCs to group 2 (n = 15), 10 µl PBS was injected into anterior chamber in group 3 (n = 10, control). Corneal opacity scoring, in vivo confocal microscopy, and histopathological evaluation were done at the end of 8 weeks. Immunofluorescence sections were evaluated to detect transplanted cells. Immune staining was performed to measure the expression levels of keratocan, aldehyde dehydrogenase (ALDH) and CD34. The gene expression levels of tumor necrosis factor (TNF-α), the interleukin 6 receptor (IL-6R), interleukin 12b (IL-12b), and transforming growth factor beta (TGF-ß1) was measured on corneas. The establishment of stem cells in the corneas of the transplanted groups was confirmed by immunofluorescence staining. The expression of keratocan, ALDH, and CD34 increased in the transplanted groups (p < 0.05). The density of keratocytes increased significantly in both transplanted groups according to the in vivo confocal microscopy data (p < 0.05). The expression of TNF-α, IL-6R, and IL-12b decreased significantly in the transplanted groups (p < 0.05). Based on our findings, we consider that allogeneic stem cells facilitate the regeneration of corneal stroma and can be a cell source for stromal repopulation in diseased cornea.

Effects of mesenchymal stem cells and VEGF on liver regeneration following major resection.

PURPOSE: The study aims to determine the effects of mesenchymal stem cell (MSC) therapy and a combination therapy of MSCs transfected with vascular endothelial growth factor (VEGF) for liver regeneration after major resection. METHODS: Thirty-eight rats were divided into four groups: group 1: control (sham operation); group 2: control (70 % hepatic resection); group 3: 70 % hepatic resection + systemically transplanted MSCs; and group 4: 70 % hepatic resection + systemically transplanted MSCs transfected with the VEGF gene. MSCs were injected via the portal vein route in study groups 3 and 4. Expression levels of VEGF, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF), hepatocyte growth factor (HGF), and augmenter of liver regeneration (ALR) were analyzed in the remnant liver tissue. We investigated the levels of angiogenic factors, VEGF-receptor, angiopoietin-1 (Angpt1) and Angpt2. Biochemical parameters of liver function in blood samples were measured and a histologic assessment of the livers was performed. The postoperative liver weight and volume of each rat were measured 14 days after surgery. RESULTS: The expression levels of all measured growth factors were significantly increased in groups 3 and 4 compared to the control groups. The levels of Angpt1 and Angpt2 correlated with levels of VEGF and thus were also significantly higher in the study groups. There were significant differences between the estimated liver weights and volumes of group 4 and the resected controls in group 2. With the exception of portal inflammation, levels of all histological parameters were observed to be higher in MSC-treated groups when compared with the resected controls in group 2. CONCLUSIONS: Transplanted stem cells and MSCs transfected with VEGF significantly accelerated many parameters of the healing process following major hepatic resection. After the injection of MSCs and VEGF-transfected MSCs into the portal vein following liver resection, they were engrafted in the liver. They increased bile duct and liver hepatocyte proliferation, and secreted many growth factors including HGF, TGFß, VEGF, PDGF, EGF, and FGF via paracrine effects. These effects support liver function, regeneration, and liver volume/weight.

Use of Adipose-Derived Mesenchymal Stem Cells to Increase Viability of Composite Grafts.

OBJECTIVE: Chondrocutaneous composite grafts figure among the reconstruction alternatives for alar rim defects resulting from tumor resection and trauma. The major problem with composite grafts is the limited graft survival area. In the present study, the authors aimed to increase the survival area of composite grafts by utilizing the ability of stem cells to promote neovascularization which is crucial in composite graft viability. METHODS: The study included 36 adult Wistar Albino rats, which were allocated to 6 groups. Groups 1, 2, and 3 were the groups in which the grafts were implanted immediately after the defect was formed, and Groups 4, 5, and 6 were those in which grafts were adapted 4 days after the defect was formed. Composite grafts of 1 × 1 cm containing both the cartilage and the skin were prepared from 1 ear, and after forming punctures and incisions on the cartilage, the grafts were adapted to the 1 × 1 cm defects on the back. The backs of the rats in groups 1 and 4 were injected with adipose-derived stem cell (ADSC), those in groups 2 and 5 with medium solution, while the rats in Groups 3 and 6 did not receive any injection. The procedures were followed by histopathological and scintigraphic evaluations. RESULTS: An evaluation of the statistical results showed that composite graft survival areas of the group treated with stem cells increased significantly, in comparison with control and medium groups. When scintigraphic evaluations were considered, it was seen that the group treated with stem cells had significantly higher radioactive substance retention than the control group. Histopathological examination demonstrated that microscopic survival rates in the stem cell group were higher than those in the control group. Green fluorescent protein (GFP) was used in the experiment to tag adipose tissue-derived stem cells. Immunofluorescence staining studies showed less apoptosis and fewer GFP (+) stem cells in the composite grafts of the stem cell group. However, apoptosis was more severe in the control and medium groups which also had decreased vascularity in the graft. DISCUSSION: As the authors have shown in the present study, ADSCs have favorable effects on the viability of composite grafts. They have increased the survival rate of the grafts to a considerable extent. As a clinical implication of this experimental study, the authors think that in the patient of auricular and nasal defects involving the cartilage and the skin, injection of the ADSC and the adaptation of composite grafts 4 days after the preparation of the receiving bed may increase the composite graft viability rates. Thus, it has been found that if the composite grafts are implanted 4 days after stem cell injection, the injection of adipose tissue-derived mesenchymal stem cells is useful in enhancing the survival of composite grafts.

Neuroprotective effects of intravitreally transplanted adipose tissue and bone marrow-derived mesenchymal stem cells in an experimental ocular hypertension model.

BACKGROUND AIMS: The purpose of this study was to investigate the neuroprotective effects of bone marrow bone marrow-derived and adipose tissue-derived mesenchymal stromal cells (MSCs) that were intravitreally transplanted in an experimental ocular hypertension (OHT) model. METHODS: An OHT rat model was generated by means of intracameral injection of hyaluronic acid into the anterior chamber. MSCs labeled with green fluorescence protein were transplanted intravitreally 1 week after OHT induction. At the end of the second and fourth weeks, retinal ganglion cells were visualized with the use of a flat-mount retina method and were evaluated by means of immunofluorescence staining against green fluorescence protein, vimentin, CD105, and cytokines (interleukin [IL]-1Ra, prostaglandin E2 receptor, IL-6, transforming growth factor-ß1, interferon-γ and tumor necrosis factor-α). RESULTS: The retinal ganglion cell numbers per area were significantly improved in stem cell-treated OHT groups compared with that in the non-treated OHT group (P < 0.05). The results of immunohistochemical analyses indicated that a limited number of stem cells had integrated into the ganglion cell layer and the inner nuclear layer. The number of cells expressing proinflammatory cytokines (interferon-γ and tumor necrosis factor-α) decreased in the MSC-transferred group compared with that in the OHT group after 4 weeks (P < 0.01). On the other hand, IL-1Ra and prostaglandin E2 receptor expressions were increased in the rat bone marrow-derived MSC group but were more significant in the rat adipose tissue-derived MSC group (P < 0.01). CONCLUSIONS: After intravitreal transplantation, MSCs showed a neuroprotective effect in the rat OHT model. Therefore, MSCs promise an alternative therapy approach for functional recovery in the treatment of glaucoma.

Comparison of Treatments With Local Mesenchymal Stem Cells and Mesenchymal Stem Cells With Increased Vascular Endothelial Growth Factor Expression on Irradiation Injury of Expanded Skin.

INTRODUCTION: Radiation injury results in chronically ischemic tissue. Radionecrosis can be encountered in severe cases. Mesenchymal stem cells (MSCs) have a therapeutic effect on ischemia-related lesions. In here, effects of bone-marrow derived MSC and vascular endothelial growth factor (VEGF) gene-transfected MSC (VEGF-MSC) treatment on expanded skin with irradiation injury is investigated. METHODS: Silicone tissue expander (50 cm) was placed subcutaneously and expanded weekly up to 60 cm in 24 Sprague Dawley rats. Single fraction (30 Gy) radiotherapy was applied to the 2 × 2 cm area of the expanded skin. Dulbecco modified Eagle medium without cell component, MSCs, and VEGF-MSCs were injected subcutaneously at the irradiation-expansion sites. Skin samples were evaluated by histomorphometry and immunohistochemistry. Perfusion rate of the samples was assessed by scintigraphy. RESULTS: Epidermal thickness of irradiated-expanded skin was increased after MSC and VEGF-MSC treatments, whereas dermal and capsule thicknesses did not change. The MSC and VEGF-MSC treatments were effective in preserving, respectively, CD31 and VEGF expressions at a similar level as expanded skin after irradiation injury. The VEGF-MSC treatment significantly elevated CD31 levels in the irradiated tissue. Skin perfusion results were consistent with the CD31 and VEGF expressions. The MSC and VEGF-MSC treatments were effective in increasing proliferating cell nuclear antigen (PCNA) expression in irradiation zone. The VEGF-MSC treatment was efficient in reducing both expansion- and irradiation-related apoptosis. CONCLUSION: Vascular impairment and dermal insufficiency due to tissue expansion and irradiation injury can easily result in a wound hard to repair. The MSCs and VEGF-MSCs can promote neovascularization, reverse the effect of irradiation, and provide more durable soft tissue for expansion/implant reconstruction.

Adipose-Derived Stem Cells Improve Survival of Random Pattern Cutaneous Flaps in Radiation Damaged Skin.

BACKGROUND: Tissue ischemia and necrosis following surgery after radiotherapy on the skin and subcutaneous tissue are well known to all reconstructive surgeons. Nevertheless, there has been no report so far on local effects of adipose-derived stem cells (ADSCs) on random flap survival elevated in an irradiated rat dorsum. In this experimental study, we aimed to identify the effect of adipose tissue-derived stem cell injection on random flap survival in irradiated tissues. METHODS: Adipose-derived stem cells were isolated from the groin region of Sprague-Dawley rats and expanded ex vivo for 3 passages. Animals were divided into 2: irradiated and nonirradiated and then again into ADSC injected and noninjected groups altogether 4 groups. After elevation of caudally based dorsal random skin flaps (10  cm long and 3  cm wide), Green fluorescent protein labeled ADSCs were then injected to the base of the pedicle. Radiotherapy was 20  Gy single dose applied during 8 weeks before surgery. At postoperative day 7, flap viability measurement and tissue harvest for histologic and immunocytochemical assessment were performed in all groups. RESULTS: We have observed increased flap viability in ADSCs injected irradiated group compared with control radiation group with small but not statistically significantly increase in vessel count per field. Mean survival rate of the flaps in groups A, B, C, and D were 40.46%, 60.07%, 40.90%, and 56.13%, respectively. There was a statistically significant vessel count difference between group B and group A and also with group D (P < 0.001). CONCLUSIONS: These findings suggest that ADSCs have a potential for enhancing the blood supply of random pattern skin flaps after radiation injury. This mechanism might be both neovascularization and vasodilation along with endothelial repair. Further studies are needed.

Comparison of aquaporin profile of advanced passage mesenchymal stem cells with early passage mesenchymal stem cells and determination of its effect on adipogenic differentiation efficiency.

OBJECTIVE: Our study aimed to compare aquaporin profiles in advanced and early passage bone marrow-derived mesenchymal stem cells (BM-MSCs) and assess the impact of aquaporin changes after adipogenic differentiation. Aquaporins are crucial for stem cell survival and differentiation during their life cycle. We focused on the role of aquaporins in the cell structures of advanced and early passage stem cells. METHODS: In our study, BM-MSCs were used for our objectives. Characterization of the cells was evaluated via flow cytometry using stem cell surface markers. The characterized BM-MSCs were divided into control and differentiation groups at passages 3 (P3) and 8 (P8). AQP1, AQP3, AQP7, AQP9, and AQP10 expression levels on days 0, 1, 3, 7, 14, and 21 were evaluated using Real Time-PCR, ELISA, and immunofluorescence studies. RESULTS: The cells were characterized by flow cytometry and confirmed to exhibit BM-MSC characteristics. At P3 and P8, differentiation was initiated, and AQP protein expression was observed to initially increase and then decrease on subsequent days. The increase in AQP protein expression at P3 occurred earlier than that at P8. Gene expression analysis demonstrated a statistically significant increase in AQP gene expression on days when AQP protein expression decreased. Moreover, statistical differences were observed between late and early passage AQP profiles. CONCLUSION: Our study examined the composition of AQPs in BM-MSCs in association with cell passage, and found that AQPs play a role in the differentiation process. The connection between the AQP profile and aging might be related to differentiation capacity, which could have implications for slowing down cellular aging and developing new therapeutic approaches.

Different Effect of Dienogest on Endometrium Mesenchymal Stem Cells Derived from Healthy and Endometriosis Tissues.

Background: Endometriosis (EM) is an inflammatory condition in which the endometrium is observed to develop outside the uterine cavity. Endometrium has conventionally been recognized as a rich source of endometrial mesenchymal stem cells (E-MSCs). Due to their secretion of proteins and other factors that modulate tissue function, E-MSCs are essential for tissue balance and repair in regenerative medicine. The influence of dienogest, a medication frequently prescribed for EM, on E-MSCs has not been extensively investigated. Aims: To explore effects of dienogest on the E-MSCs derived from healthy (E-MSCs-control) and diseased (E-MSCs-endometriosis) endometrial tissue samples in vitro. The main outcome is that dienogest-based treatments might also be designed to target stem cells in endometrial tissues, which enables it to effectively regulate disease progression. Study Design: In vitro study. Methods: We collected samples from healthy and diseased endometrial tissues. E-MSCs were derived from both healthy and EM tissues. The effect of dienogest (VISANNE) on E-MSCs was assessed by examining cell proliferation, telomerase activity, cell migration, and estrogen secretion levels after the isolation and characterization of E-MSCs. Results: We discovered that cellular proliferation rate was higher in the E-MSCs derived from EM tissues compared to those derived from healthy tissue. The proliferation rate and telomerase activity were both suppressed by dienogest treatment, particularly in E-MSCs-endometriosis. The drug treatment also resulted in a decrease in the migration capacity of E-MSCs-endometriosis, from 60.4% to 59.2%. The expression of CXCL12, Ki67, and beta-catenin was analyzed in both E-MSCs-endometriosis and E-MSCs-control. The CXCL12 and Ki67 expressions were quite elevated in the E-MSCs-endometriosis without drug treatment compared to the E-MSCs-control. Following the treatment, these levels declined drastically to the levels close to E-MSCs-control. Similarly, this decrease in gene expression was accompanied by a decrease in estrogen secretion into the medium. Conclusion: This research demonstrates that dienogest exerts a substantial impact on both stromal and stem cells, as it effectively controls the disease by reversing EM markers, despite the absence of progesterone receptors on endometrial stem cells.

Production of alginate macrocapsule device for long-term normoglycaemia in the treatment of type 1 diabetes mellitus with pancreatic cell sheet engineering.

Type 1 diabetes-mellitus (T1DM) is characterized by damage of beta cells in pancreatic islets. Cell-sheet engineering, one of the newest therapeutic approaches, has also been used to create functional islet systems by creating islet/beta cell-sheets and transferring these systems to areas that require minimally invasive intervention, such as extrahepatic areas. Since islets, beta cells, and pancreas transplants are allogeneic, immune problems such as tissue rejection occur after treatment, and patients become insulin dependent again. In this study, we aimed to design the most suitable cell-sheet treatment method and macrocapsule-device that could provide long-term normoglycemia in rats. Firstly, mesenchymal stem cells (MSCs) and beta cells were co-cultured in a temperature-responsive culture dish to obtain a cell-sheet and then the cell-sheets macroencapsulated using different concentrations of alginate. The mechanical properties and pore sizes of the macrocapsule-device were characterized. The viability and activity of cell-sheets in the macrocapsule were evaluatedin vitroandin vivo. Fasting blood glucose levels, body weight, and serum insulin & C-peptide levels were evaluated after transplantation in diabetic-rats. After the transplantation, the blood glucose level at 225 mg dl-1on the 10th day dropped to 168 mg dl-1on the 15th day, and remained at the normoglycemic level for 210 days. In this study, an alginate macrocapsule-device was successfully developed to protect cell-sheets from immune attacks after transplantation. The results of our study provide the basis for future animal and human studies in which this method can be used to provide long-term cellular therapy in T1DM patients.

Sublingual Administration of Teucrium Polium-Loaded Nanofibers with Ultra-Fast Release in the Treatment of Diabetes Mellitus: In Vitro and In Vivo Evaluation.

In this study, Teucrium polium (TP) methanolic extract, which has antidiabetic activity and protects the ß-cells of the pancreas, was loaded in polyethylene oxide/sodium alginate nanofibers by electrospinning and administered sublingually to evaluate their effectiveness in type-2 diabetes mellitus (T2DM) by cell culture and in vivo studies. The gene expressions of insulin, glucokinase, GLUT-1, and GLUT-2 improved in TP-loaded nanofibers (TPF) on human beta cells 1.1B4 and rat beta cells BRIN-BD11. Fast-dissolving (<120 s) sublingual TPF exhibited better sustainable anti-diabetic activity than the suspension form, even in the twenty times lower dosage in streptozotocin/nicotinamide-induced T2DM rats. The levels of GLP-1, GLUT-2, SGLT-2, PPAR-γ, insulin, and tumor necrosis factor-alpha were improved. TP and TPF treatments ameliorated morphological changes in the liver, pancreas, and kidney. The fiber diameter increased, tensile strength decreased, and the working temperature range enlarged by loading TP in fibers. Thus, TPF has proven to be a novel supportive treatment approach for T2DM with the features of being non-toxic, easy to use, and effective.

Endoplasmic reticulum stress of endometrial mesenchymal stem cells in endometriosis.

OBJECTIVE: The human endometrium has significant regenerative abilities due to stem cells, which are vital in immunomodulation, immune tolerance, steroid hormone response, and inflammation. Endometriosis, an inflammatory gynecological disorder where endometrium-like tissue grows outside uterus, affects millions of women and often causes infertility. Recent research indicates that stem cells contribute to pathology of endometriosis. ER stress is implicated in various diseases, including endometriosis. This study aims to examine ER stress in eMSCs within endometriosis pathogenesis and uncover underlying disease mechanisms. METHODS: Samples were collected from healthy subjects and women with endometriosis in both proliferative and secretory phases. eMSCs were isolated and characterized via flow cytometry. ER stress protein levels were assessed using proteomic analysis, with validation through Western Blot and immunofluorescence staining. Gene expression was analyzed by RT-qPCR, and ultrastructural examination of eMSCs was conducted using TEM. ER stress markers in tissue samples were detected in SUSD2+ eMSCs through immunofluorescence staining and visualized using a confocal microscope. Statistical analysis was performed using SPSS program. RESULTS: The proteomics analysis uncovered ER stress-related proteins (DDRGK1, RTN3, ERp44, TMED2, TMEM33, TMX3) whose levels were significantly distinct from control group. Western Blot analysis and immunofluorescence staining results at protein level; RT-qPCR results at gene level supported these findings. TEM analysis also showed ultrastructural presence of ER stress in endometriosis groups. CONCLUSION: Presence of ER stress in eMSCs in pathogenesis of endometriosis has been demonstrated using various methods. Our research has potential to shed light on pathology of endometriosis and offer promising avenues for non-invasive diagnosis and potential treatment.

Investigation of the differentiation potential of pericyte cells as an alternative source of mesenchymal stem cells.

BACKGROUND: The mesenchymal stem cells (MSCs) with characterized by their multipotency and capacity to differentiate into various tissue cell types, have led to their incorporation in regenerative medicine research. However, the limited numbers of MSCs in the human body and their diverse differentiation capabilities in tissues highlight the need for exploring alternative regenerative cell sources. In this study, therefore, we conducted molecular level examinations to determine whether pericytes, specialized cell communities situated near blood vessels, could serve as a substitute for human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this context, the potential application of pericytes surrounds the vessels when MSCs are insufficient for functional purposes. METHODS: The pericytes utilized in this investigation were derived from the placenta and characterized at the third passage. Similarly, the hBM-MSCs were also characterized at the third passage. The pluripotent properties of the two cell types were assessed at the gene expression level. Thereafter, both pericytes and hBM-MSCs were directed towards adipogenic, osteogenic and chondrogenic differentiation. The cells in both groups were examined on days 7, 14, and, 21 and their differentiation status was compared both immunohistochemically and through gene expression analysis. RESULTS: Upon comparing the pluripotency characteristics of placental pericytes and hBM-MSCs, it was discovered that there was a substantial upregulation of the pluripotency genes FoxD3, Sox2, ZPF42, UTF1, and, Lin28 in both cell types. However, no significant expression of the genes Msx1, Nr6a1, Pdx1, and, GATA6 was observed in either cell type. It was also noted that pericytes differentiate into adipogenic, osteogenic and, chondrogenic lineages similar to hBM-MSCs. DISCUSSION: As a result, it has been determined that pericytes exhibit high differentiation and proliferation properties similar to those of MSCs, and therefore can be considered a suitable alternative cell source for regenerative medicine and tissue engineering research, in cases where MSCs are not available or insufficient. It is notable that pericytes have been suggested as a potential substitute in studies where MSCs are lacking.

Adipose tissue-derived mesenchymal stromal cells efficiently differentiate into insulin-producing cells in pancreatic islet microenvironment both in vitro and in vivo.

BACKGROUND AIMS: Differentiation or reprogramming of stem cells could be achieved by remodulating the microenvironment, which regulates the fate of cells by soluble factors and contacts. By providing an in vivo-like microenvironment, directional and functional differentiation of stem cells could be achieved in vitro. In this study, the differentiation of mesenchymal stromal cells (MSCs) derived from rat tissues (adipose, rAT; bone marrow, rBM) were analyzed by in vitro and in vivo co-culture experiments. The insulin-producing capacities of islets transplanted under the renal kidney capsule with rAT- and rBM-MSCs were compared and the reduction of hyperglycemia symptoms in rat models was examined. METHODS: MSCs prelabeled with green fluorescence protein were co-cultured with islets directly. The insulin production of cells was determined by immunostaining and ELISA. Streptozotocin-induced diabetic rat models were created and MSCs were co-transplanted with the islets under the kidney capsule to confirm the in vitro results. RESULTS: MSCs were differentiated into insulin-producing cells after 38 days of co-culture, confirmed by insulin and C-peptide stainings. In vivo functional studies revealed that the co-culture of islets with MSCs provided higher differentiation efficiency. The weight gain measurement and glucose tolerance test in the rat group co-transplanted of rAT-MSCs and islets indicate a better recovery than islet-alone transplants and co-transplants of islets and rBM-MSCs. CONCLUSIONS: rAT-MSCs could be considered as the cell of choice for cell-based treatment of type 1 diabetes. Because the co-transplantation of islets with MSCs increases the number of insulin-producing cells, this method was suggested for clinical applications.

Genomagnetic assay for electrochemical detection of osteogenic differentiation in mesenchymal stem cells.

The osteogenic differentiation of mesenchymal stem cells (MSCs) was assessed by determining the gene expression levels of proteins; osteocalcin (OSC), osteonectin (OSN) and osteopontin (OSP) based on electrochemical detection protocol combined with genomagnetic assay in parallel to real-time PCR analysis. Genomagnetic assay was performed using streptavidin coated commercial magnetic particles (magnetic beads, MBs) in combination with single-use electrochemical sensor technology. A biotinylated DNA probe was immobilized onto streptavidin coated magnetic particles, and then the hybridization process of the probe with its complementary DNA was performed. The oxidation signals of DNA electroactive bases guanine and adenine were measured voltammetrically using a pencil graphite electrode (PGE) before and after the hybridization process of OSC/OSN/OSP probe sequences with their complementary target sequences. The selectivity of the genomagnetic assay was also tested using each DNA probe individually related to osteogenic differentiations. The voltammetric detection of osteogenic differentiations was confirmed selectively by real-time PCR analysis.

Propolis prevents vascular endothelial dysfunction by attenuating inflammation and oxidative damage in the chronic unpredictable stress model of depression in rats.

OBJECTIVES: Chronic stress may lead to depression and vascular endothelial dysfunction. We aimed to evaluate the effects of propolis on vascular functions and the possible mechanisms of its vascular effects in the rat model of chronic unpredictable mild stress (CUMS)-induced depression. METHODS: Male Wistar rats were divided into control, stress (exposure to CUMS), control+propolis and stress+propolis groups (n = 8/each group). CUMS model was induced by exposing rats to various mild stressors daily for 5 weeks. The extract of propolis (100 mg/kg/day) was administered orally to propolis-treated groups for 5 weeks. The depression-like behaviours were assessed with the forced swimming test (FST). Chronic stress resulted in increased immobility response in FST and elevated serum corticosterone levels. Thoracic endothelial functions and expressions of endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha (TNFα), interleukin-1beta (IL-1ß), Heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) level were assessed. KEY FINDINGS: Compared to control group, stress group exhibited a significant decrease in endothelium-dependent relaxations, and eNOS, SOD and HO-1 expressions, whereas a significant increase in the thoracic expressions of TNFα and IL-1ß. Propolis ameliorated depression-like behaviours, vascular endothelial dysfunctions and alterations of protein expressions. CONCLUSION: Propolis exerted antidepressant-like and vasculoprotective effects in CUMS-induced depression in rats. Chronic propolis treatment may have a protective effect on CUMS-induced vascular endothelial dysfunction by its anti-inflammatory and antioxidant effects.