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.

Examining the effect of activated cytotoxic (CD8+) T-cell exosomes to the lung cancer.

Lung cancer continues to be a major health problem worldwide owing to its incidence, and causes physical, psychological, social, and economic problems. Activated cytotoxic T cells (ACTC) are positively correlated with the tumor microenvironment (TME), improving the prognosis of cancer patients. Recently, ACTC-derived exosomes (ACTC-dExo) were implicated in this effect by inhibiting mesenchymal stem cells, which may promote metastasis in the TME. Exosomes are thought to be advantageous for the specific delivery of drugs to cancer cells because they have the characteristics of natural liposomes, are nanosized, and remain largely stable in the blood due to the protein and lipid content they carry on their membranes. In this study, we aimed to determine the cytotoxic and metastatic inhibitory effects of ACTC-dExo in A549 cells in vitro. Cytotoxic CD8+ T cells were isolated from whole blood obtained from healthy individuals and cultured for 5-7 days after stimulation. The ACTC-dExo serum-free culture medium was collected by ultracentrifugation. Characterization and quantification of the isolated exosomes were performed using flow cytometry, electron microscopy, zeta-sizer measurements, and bicinchoninic acid (BCA) assays. We co-cultured ACTC and ACTC-dExo with A549 cells for 48 h. The viability of A549 cells was evaluated using a WST-1 assay. The metastasis-related genes MMP2, MMP9, TWIST, SNAI1, and CDH1 were detected by qRT-PCR, and MMP2 and MMP9 proteins were evaluated by confocal microscopy. In addition, changes in cell migration were investigated using a scratch assay. ACTC-dExo were found to have anti-proliferative and anti-metastatic effects and reduced cancer cell proliferation and metastatic properties.

Investigation of impacts of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of mesenchymal stem cell through Notch/Hedgehog signaling pathways.

OBJECTIVE: Decellularization is the process to obtain natural scaffolds with tissue integrity and extracellular matrix components, and recellularization is used to produce tissue-like constructs with specific cell types. In this study, rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) were cultured on decellularized heart extracellular matrix. These cells were then induced to differentiate into cardiomyogenic cells under the stimulatory effect of vascular endothelial growth factor (VEGF) and other chemicals. This study aimed to investigate the effect of the cardiac extracellular matrix and VEGF on cardiomyogenic differentiation in the context of the Notch and Hedgehog signaling pathways. METHODS: Heart samples extracted from rats were decellularized by serial application of detergent to remove cells from the tissue, and then recellularized with rBM-MSCs. The recellularized tissue matrices were then analyzed for cardiomyogenesis. Cardiomyogenic differentiation was performed on decellularized heart extracellular matrix (ECM; three-dimensional scaffolds) and culture plates (two-dimensional cell culture system) for 28 days to understand the effects of the heart extracellular matrix. In addition, differentiation was induced with and without the stimulatory effect of VEGF to understand the effect of VEGF on cardiomyogenic differentiation of rBM-MSCs. RESULTS: Immunofluorescence staining showed that decellularization of the heart was performed effectively and successfully. After decellularization process, the heart extracellular matrix was completely free of cells. It was observed that rBM-MSCs transplanted onto the heart extracellular matrix remained viable and proliferated for 21 days after recellularization. The rBM-MSCs promoted cardiomyogenic differentiation in the conventional differentiation medium but were inversely affected by both VEGF and heart extracellular matrix proteins. Lower expression of connexin43 and cardiac troponin I genes was observed in cells induced by either matrix proteins or VEGF, compared to cells differentiated by chemical agents alone. CONCLUSION: In this study, we investigated the effect of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of rBM-MSCs. On the decellularized cardiac extracellular matrix, rBM-MSCs maintained their viability by adhering to the matrix and proliferating further. The adhesion of the cells to the matrix also produced a physical stimulus that led to the formation of histological structures resembling myocardial layers. Chemical stimulation of the decellularized heart extracellular matrix and cardiomyogenic differentiation supplements resulted in increased expression of cardiomyogenic biomarkers through modulation of the Notch and Hedgehog signaling pathways.

Etanercept Prevents Endothelial Dysfunction in Cafeteria Diet-Fed Rats.

Obesity is associated with endothelial dysfunction and this relationship is probably mediated in part by inflammation. Objective: The current study evaluated the effects of etanercept, a tumor necrosis factor-alpha (TNF-α) inhibitor, on endothelial and vascular reactivity, endothelial nitric oxide synthase (eNOS) immunoreactivity, and serum and aortic concentrations of TNF-α in a diet-induced rat model. Design and results: Male weanling Wistar rats were exposed to a standard diet and cafeteria diet (CD) for 12 weeks and etanercept was administered during CD treatment. Isolated aortas of the rats were used for isometric tension recording. Carbachol-induced relaxant responses were impaired in CD-fed rats, while etanercept treatment improved these endothelium-dependent relaxations. No significant change was observed in papaverine- and sodium nitroprusside (SNP)-induced relaxant responses. eNOS expression decreased in CD-fed rats, but no change was observed between etanercept-treated CD-fed rats and control rats. CD significantly increased both the serum and the aortic levels of TNF-α, while etanercept treatment suppressed these elevated levels. CD resulted in a significant increase in the body weight of the rats. Etanercept-treated (ETA) CD-fed rats gained less weight than both CD-fed and control rats.

THE EFFECTS OF ETANERCEPT AND CABERGOLINE ON ENDOMETRIOTIC IMPLANTS, UTERUS AND OVARIES IN RAT ENDOMETRIOSIS MODEL.

The pathophysiology of endometriosis is still unknown and treatment options remain controversial. Searches focus on angiogenesis, stem cells, immunologic and inflammatory factors. This study investigated the effects of etanercept and cabergoline on ovaries, ectopic, and eutopic endometrium in an endometriosis rat model. This randomized, placebo-controlled, blinded study included 50 rats, Co(control), Sh(Sham), Cb(cabergoline), E(etanercept), and E + Cb(etanercept + cabergoline) groups. After surgical induction of endometriosis, 2nd operation was performed for endometriotic volume and AMH level. After 15 days of treatment: AMH level, flow cytometry, implant volume, histologic scores, immunohistochemical staining of ectopic, eutopic endometrium, and ovary were evaluated at 3rd operation. All groups had significantly reduced volume, TNF-α, VEGF, and CD 146/PDGF-Rß staining of endometriotic implants comparing to the Sh group (p < 0.05).TNF-α staining of eutopic endometrium in all treatment groups was similar to Sh and Co groups (p > 0.05). E and E + Cb groups significantly decreased TNF-α staining in the ovary comparing to Sh, Co, and Cb groups (p < 0.05). All treatment groups had significantly higher AFC compared to the Sh group. CD25+ Cells' median percentage was significantly increased in the E + Cb group compared to Co, Sh, Cb, and E group. E + Cb group had a significantly higher CD5+ Cells' level than the Co group (p = 0.035). In conclusion; Etanercept and/or Cabergoline decreased volume, TNF-α, VEGF, and CD 146/PDGF-Rß staining of the ectopic endometrial implant. E and E + Cb treatment decreased TNF-α levels in the ovary. E + Cb also increased peripheral blood CD25+ & CD5+ Cell's.

Depression induced by chronic stress leads to penile cavernosal dysfunction: protective effect of anti-TNF-α treatment.

Psychological stress may lead to erectile dysfunction (ED), and inflammation has been evaluated as a major contributing factor. The goal of this study was to investigate the effects of etanercept (ETN), an anti-tumor necrosis factor α (TNF-α) protein, on cavernosal function in the unpredictable chronic mild stress (UCMS) rat model of depression. Animals were divided into 4 groups: animals not exposed to UCMS, animals not exposed to UCMS and treated with ETN, animals exposed to UCMS, and animals treated with ETN while exposed to UCMS. UCMS significantly impaired the neurogenic and endothelium-dependent relaxation responses; reduced cavernosal endothelial nitric oxide (NO) synthase (eNOS) and neuronal NO synthase (nNOS) expressions; decreased testosterone levels; enhanced systemic levels of corticosterone, TNF-α, interleukin 1ß (IL-1ß), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule 1 (ICAM-1); and also increased cavernosal levels of TNF-α, IL-1ß, and IL-6 in rats. ETN administration restored NO-mediated neurogenic and endothelium-dependent relaxation responses of the corpus cavernosum, increased cavernosal eNOS and nNOS expressions, enhanced testosterone levels, and decreased corticosterone levels in UCMS-exposed rats. Also, systemic inflammatory markers and cavernosal proinflammatory cytokine levels were reduced by ETN. Our results demonstrate the role of TNF-α-mediated inflammation in the development of depression and ED in rats exposed to chronic stress.

TNF-α antagonism with etanercept enhances penile NOS expression, cavernosal reactivity, and testosterone levels in aged rats.

Erectile dysfunction (ED) has been reported to be associated with inflammation. This study investigated the effects of tumor necrosis factor alpha (TNF-α) inhibitor etanercept on penile neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) expressions, testosterone concentrations, neurogenic and endothelium-dependent relaxations of corpus cavernosum (CC), and circulating and cavernosal levels of inflammatory markers in aged rats. Animals were separated into control, aged, and etanercept-treated aged groups. Aged rats displayed significantly increased serum and cavernosal TNF-α, C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule (ICAM-1) levels, and decreased penile nNOS and eNOS expressions and serum testosterone levels compared with controls. In etanercept-treated aged group, NOS expressions were similar to that of the control group. The circulating and cavernosal concentrations of TNF-α, CRP, MCP-1, ICAM-1, and testosterone were also normalized by etanercept. Neurogenic and endothelium-dependent relaxant responses significantly decreased in aged rats and etanercept treatment markedly improved these relaxation responses. Our findings indicate that aging decreases penile NOS expression, neurogenic and endothelium-dependent relaxations of CC, and also suppresses serum testosterone levels by inducing inflammatory response that may contribute to the development of ED. TNF-α antagonism may be a novel strategy to treat aging-associated ED.

The distribution of CD44+/CD24- cancer stem cells in breast cancer and its relationship with prognostic factors.

PURPOSE: The purpose of this study was to investigate the correlation between the percentages of CD44+/CD24- cancer stem cells (CSCs) and the clinicopathological and prognostic factors in breast cancer patients. METHODS: Twenty three women who underwent surgery for breast cancer were enrolled in this study. The mean age of the patients was 46.65 years and 52% had early-stage disease. Tumor tissues obtained during surgery were digested enzymatically. CD44+/CD24- cell phenotype was identified by using surface marker antibodies and percentages were determined by surface marker expression of the cells. RESULTS: Sixty five percent of the tumors were positive for estrogen (ER)/ progesterone receptors (PR) and 38% of the tumors were positive for HER-2. All of the patients with hormone receptor positive tumors had ER positive tumors, while only 11 patients had PR positive breast cancer. CD44+/CD24- cells were present in all tumor tissues. The mean proportion of the CD44+/CD24- cells was 1.43±1.6. The mean percentages of CD18+ cells and MUC1+ were 27.9±26.5% and 6.07±11.34%, respectively. The percentage of CD18+ cells was significantly higher in PR positive tumors (p=0.042). There was no significant correlation between the percentage of CD44+/CD24- cells and clinicopathological features. CONCLUSION: This study showed that CD44+/CD24- cells were present in all tumor tissues. The percentage of CD44+/CD24- cells was higher in early-stage disease, yet without statistical significance. No correlation was found between prognostic factors and the percentage of the CD44+/CD24- cells.

Protective effects of resveratrol on aging-induced cognitive impairment in rats.

Resveratrol, a polyphenol phytoalexine, has been shown to play a neuroprotective role in the neurodegenerative process in Alzheimer's disease (AD) and improve memory function in dementia. However, the in vivo effect of resveratrol in normal aging models of learning and memory has not yet been evaluated. Therefore, the present neurobehavioral study was undertaken to evaluate the effect of resveratrol on cognitive impairment induced by aging in passive avoidance and Morris water maze (MWM) tests. Male Wistar albino rats were divided into four groups: young control (4month), young resveratrol (4month+RESV), old control (24month) and old resveratrol (24month+RESV). Resveratrol (50mg/kg/day) was given to the 4month+RESV and 24month+RESV groups orally for 12weeks. There was no significant difference between the groups for the first day of latency, while in aged rats, the second day of latency was significantly shortened compared to the young group in the passive avoidance test (p<0.05). Additionally, in the MWM test, the results showed a decrease in the time spent in the escape platform's quadrant in the probe test in aged rats (p<0.05). The administration of resveratrol at 50mg/kg/day increased the retention scores in the passive avoidance test and the time spent in the escape platform's quadrant in the MWM task (p<0.05). Furthermore resveratrol attenuated the protein levels of TNFα and IL1ß in the 24-month group. These findings indicate that aging impairs emotional and spatial learning-memory and resveratrol reverses the effect of age-related learning and memory impairment. The results of this study suggest that resveratrol is effective in preventing cognitive deficit in aged rats by inhibiting the production of inflammatory cytokines.

Cross effects of resveratrol and mesenchymal stem cells on liver regeneration and homing in partially hepatectomized rats.

In this study, we examined the effect of preoperatively administered resveratrol (RV) and mesenchymal stem cells (MSCs) on regeneration of partially hepatectomized rat liver. We also evaluated the effect of RV on homing of MSCs. MSCs were isolated from bone marrow and cultured in vitro. Wistar albino rats were randomly divided into four groups. In groups, rats received (1) no treatment, (2) single dose RV, (3) MSCs and (4) RV plus MSCs before partial hepatectomy (PH). Injected MSCs were traced by labeling them with green fluorescent protein, and liver regeneration was determined by comparison of liver weight gain, histological examination and immunohistochemical staining with proliferating cell nuclear antigen (PCNA) for mitotic cells. The expression levels of tumor necrosis factor -alpha (TNF-α), interleukin-6 (IL-6) and hepatocyte growth factor (HGF) were also determined in the parafin sections of liver specimens with immunohistochemical staining. Administration of RV and MSCs separately or together enhanced liver regeneration despite decreasing the TNF-α and IL-6 expression. This positive contribution was probably due to direct raising effect on HGF for RV and HGF expression for MSCs that we demonstrated with immunohistochemical staining. Additionally, RV increased the homing of MSCs in liver probably related to life prolonging effect on MSCs. These results indicate that preoperative RV as well as MSCs application enhances liver regeneration after partial hepatectomy in rats. Paying attention to RV about the effect on liver regeneration and homing of MSCs might be the goal of further investigations.

In vitro protection of adipose tissue-derived mesenchymal stem cells by erythropoietin.

Mobilization of stem cells and their differentiation into cardiomyocytes are known to have protective effects after myocardial infarction. The integrity of transplanted mesenchymal stem cells for cardiac regeneration is dependent on cell-cell or cell-matrix interaction, which is adversely affected by reactive oxygen species in an ischemic environment. Treatment with erythropoietin was shown to protect human adipose tissue derived mesenchymal stem cells in an ischemic injury in vitro model. The analyses indicated that expression of erythropoietin receptors played a pivotal role in erythropoietin mediated cell survival. In this study, the anti-apoptotic effect of erythropoietin on stem cells was analyzed in apoptosis-induced human mesenchymal stem cells. Apoptosis was induced in cultured adult human adipose tissue derived mesenchymal stem cells by hydrogen peroxide. A group of cultured cells was also treated with recombinant human erythropoietin in a concentration of 50 ng mL(-1). The degree of apoptosis was analyzed by flow-cytometry and immunohistochemical staining for Caspase 3. The average percentages of apoptotic cells were significantly higher in H2O2-induced stem cells than in cells co-cultured with erythropoietin (63.03 ± 4.96% vs 29 ± 3.41%, p<0.01). We conclude that preconditioning with erythropoietin suppresses apoptosis of mesenchymal stem cells and enhances their survival.

The short- and long- term effects of estrogen deficiency on apoptosis in musculoskeletal tissues: an experimental animal model study.

BACKGROUND: Estrogen is the major sex steroid affecting the growth, remodeling, and homeostasis of the female skeleton. Estrogen loss in postmenopausal women leads to osteoporosis. The aim of this study was to evaluate the early and long- term effects of estrogen loss on bones, tendons, muscles, and menisci in ovariectomized rats.  METHODS: Fifteen rats were randomized into three groups of five animals each. The first group was the control group with no additional surgical procedure, but the rest (groups 2 and 3) were bilaterally ovariectomized . All animals in the group 2 were sacrificed at 14th week to evaluate the short- term effect, and all of other animals in the groups 1 and 3 were sacrificed at 28th week to analyze the long- term effect of estrogen loss in the ovariectomized group and to control with the group 1. Quadriceps muscles, Achilles tendons, menisci, and femur cortical bones from both lower extremities were taken. The amount of apoptosis was measured. RESULTS: There was a significant increase in cell apoptosis in bones, muscles, and tendons with insignificant increase in cell apoptosis in menisci at early and late periods in rats with ovariectomies than the control.  CONCLUSION: The results indicated that estrogen loss after ovariectomy does not only affect bones; it may also increase cell apoptosis in different tissues such as muscles, tendons, and menisci.

Comparative analysis of apoptotic resistance of mesenchymal stem cells isolated from human bone marrow and adipose tissue.

AIM: Mesenchymal stem cells (MSCs) isolated from human bone marrow (hBM) and adipose tissue (hAT) are perceived as attractive sources of stem cells for cell therapy. The aim of this study was to compare MSCs from hBM and hAT for their immunocytochemistry staining and resistance to in vitro apoptosis. METHODS: In our study, we investigated the antiapoptotic ability of these MSCs toward oxidative stress induced by hydrogen peroxide (H(2)O(2)) and serum deprivation. Results were assessed by MTT and flow cytometry. All experiments were repeated a minimum of three times. RESULTS: Flow cytometry and MTT analysis revealed that hAT-MSCs exhibited a higher resistance toward H(2)O(2)-induced apoptosis (n = 3, hBM-hAT viability H(2)O(2) 58.43 ± 1.24-73.02 ± 1.44, P < 0.02) and to serum-deprivation-induced apoptosis at days 1 and 4 than the hBM-MSCs (n = 3, hAT-hBM absorbance, resp., day 1: 0.305 ± 0.027-0.234 ± 0.015, P = 0.029, day 4: 0.355 ± 0.003-0.318 ± 0.007, P = 0.001, and day 7: 0.400 ± 0.017-0.356 ± 0.008, P = 0.672). hAT-MSCs showed superior tolerance to oxidative stress triggered by 2 mmol/L H(2)O(2) and also have superior antiapoptosis capacity toward serum-free culture. CONCLUSION: In this study we found that hAT-MSCs are more resistant to in vitro apoptosis.

Effects of resveratrol on ileal smooth muscle reactivity in polymicrobial sepsis model.

OBJECTIVE: To determine the effects of resveratrol on the ileal smooth muscle reactivity in polymicrobial sepsis. MATERIAL AND METHODS: Polimicrobial sepsis was induced by the cecal ligation and perforation (CLP) procedure. Sprague Dawley rats were divided into three groups. Rats in resveratrol group received resveratrol after CLP (100 mg/kg, i.p.). Rats received saline immediately after CLP in the sepsis group. Control group rats underwent sham operation. The rats were sacrificed and the ileum was excised 24 h after the operation. Contractile and relaxant responses in isolated smooth muscle strips (SMS) were determined using an in vitro muscle technique. TNFα and IL-6 levels were measured in blood samples. RESULTS: Contractile responses to carbachol and KCl and relaxant responses to transmural electrical field stimulation (EFS) were significantly decreased in the sepsis group compared with control and resveratrol groups. No significant changes were observed for smooth muscle reactivity in the resveratrol and control groups. Sodium nitroprusside (SNP) or papaverine-induced relaxations were similar in the all groups. Resveratrol treatment supressed increased TNFα and IL-6 levels in blood seen in sepsis group. CONCLUSION: Ileal smooth muscle reactivity was improved after resveratrol treatment in rats with sepsis. The results of the present study indicate that the beneficial effects of resveratrol might be, at least in part, attributed to its effects on non-adrenergic non-cholinergic pathway and/or anti-inflammatory and antioxidant activity.

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.

A comprehensive characterization study of human bone marrow mscs with an emphasis on molecular and ultrastructural properties.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) continue to draw attention of researchers in the fields of basic science and medicine due to their indispensible regenerative, reparative, angiogenic, anti-apoptotic, and immunosuppressive properties, all of which collectively point out their enormous therapeutic potential. There is still, however, a need for further investigation of their characteristics to broaden their field of use and learn much more about how to control their fate and improve their therapeutic effectiveness. hBM-MSCs were extensively characterized in terms of their growth characteristics, genetic stability, and differentiation capability to the mesodermal and ectodermal cell lineages; a special emphasis was given to their phenotypic and ultrastructural properties. Expression of embryonic stem cell markers Oct4, Rex-1, FoxD-3, Sox2, and Nanog was shown with real-time PCR. Transmission electron microscopy revealed the ultrastructural characteristics of hBM-MSCs; they had pale, irregularly shaped and large euchromatic nuclei, and two distinct areas in their cytoplasm: an intensely stained inner zone rich in mitochondria and rough endoplasmic reticulum (rER) with dilated cisternae and a relatively peripheral zone poor in organelles. hBM-MSCs expressed adipogenic (adipophilin and PPARγ), myogenic (desmin, myogenin, α-SMA), neurogenic (γ-enolase, MAP2a,b, c-fos, nestin, NF-H, NF-L, GFAP, ß3-tubulin), osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, type I collagen), and chondrogenic (type II collagen, SOX9) markers either at RNA or protein level even under basal conditions, without any stimulation towards differentiation. The differentiation potential of hBM-MSCs to adipogenic, osteogenic, and neurogenic lineages was shown by using the relevant differentiation factors.

Isolation and characterization of stem cells from pancreatic islet: pluripotency, differentiation potential and ultrastructural characteristics.

BACKGROUND AIMS: Stem cells (SC) in different locations have individual characteristics. Important questions to be answered include how these specialties are generated, what the mechanism underlying their generation is, and what their biologic and clinical merits are. A basic approach to answering these questions is to make comparisons between the differences and similarities among the various SC types. They may focus on aspects of biologic marker discovery, capacity of proliferation and differentiation, along with other characteristics. The aim of this study was to characterize in detail the SC isolated from pancreatic islet (PI) and compare their properties with bone marrow (BM)-derived mesenchymal stromal cells (MSC) of the rat. METHODS: Immunophenotypic characteristics, proliferation capacities, telomerase activities, pluripotent-related gene expressions, ultrastructure and the potential for multilineage differentiation of PI SC and BM MSC were studied. RESULTS: We found that PI SC expressed markers of embryonic SC (Oct-4, Sox-2 and Rex-1) and had a high proliferation capacity, proven also by high telomerase activities. Surprisingly, markers belonging to differentiated cells were expressed by these cells in a constitutive manner. PI SC ultrastructure showed more developed and metabolically active cells. CONCLUSIONS: The immunocytochemical identification of both PI SC and BM MSC was demonstrated to be typical MSC. Without stimulation of differentiation markers of adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells in these SC, the expression of those markers might explain their multilineage differentiation potential. We suggest that, by reason of the respectively high telomerase activity in PI SC, they could be better candidates than BM MSC for cell replacement therapy of type 1 diabetes.