MicroRNA-Transcription factor regulatory networks in the early strobilar development of Echinococcus granulosus protoscoleces.

BACKGROUND: Echinococcus granulosus sensu lato has a complex developmental biology with a variety of factors relating to both intermediate and final hosts. To achieve maximum parasite adaptability, the development of the cestode is dependent on essential changes in transcript regulation. Transcription factors (TFs) and miRNAs are known as master regulators that affect the expression of downstream genes through a wide range of metabolic and signaling pathways. In this study, we aimed to develop a regulatory miRNA-Transcription factor (miRNA-TF) network across early developmental stages of E. granulosus protoscoleces by performing in silico analysis, and to experimentally validate TFs expression in protoscoleces obtained from in vitro culture, and from in vivo experiments. RESULTS: We obtained list of 394 unique E. granulosus TFs and matched them with 818 differentially expressed genes which identified 41 predicted TFs with differential expression. These TFs were used to predict the potential targets of 31 differentially expressed miRNAs. As a result, eight miRNAs and eight TFs were found, and the predicted network was constructed using Cytoscape. At least four miRNAs (egr-miR-124a, egr-miR-124b-3p, egr-miR-745-3p, and egr-miR-87-3p) and their corresponding differentially expressed TFs (Zinc finger protein 45, Early growth response protein 3, Ecdysone induced protein 78c and ETS transcription factor elf 2) were highlighted in this investigation. The expression of predicted differentially expressed TFs obtained from in vitro and in vivo experiments, were experimentally validated by quantitative polymerase chain reaction. This confirmed findings of RNA-seq data. CONCLUSION: miRNA-TF networks presented in this study control some of the most important metabolic and signaling pathways in the development and life cycle of E. granulosus, providing a potential approach for disrupting the early hours of dog infection and preventing the development of the helminth in the final host.

Linguatulosis in small ruminants in southeastern Iran: Epidemiological, histopathological and phylogenetic findings and its public health importance.

Linguatulosis, as a zoonotic disease, can infect most ruminants and cause accidental infections in humans. The objective of this study was to explore the epidemiological, histopathological and phylogenetic profiles of Linguatula serrata infection in sheep and goats and its public health importance during 2015-2018. Mesenteric lymph nodes (MLNs) and liver tissue of goats and sheep were selected randomly in Kerman slaughterhouse. Nymphal samples were used for DNA extraction, amplification and subsequently phylogenetic analysis using 18s rRNA and cytochrome C oxidase subunit 1 (cox1). Overall, of 828 examined livestock, 179 (42.4%) goats and 71 (17.5%) sheep were found to be infected with the nymphal stage of L. serrata. A significant difference was observed between linguatulosis and age. In the histopathological assessment, longitudinal and transverse sections of L. serrata nymphs were observed within the cyst-like spaces surrounded by a wall of fine fibrosis and compact lymphocytes. Moreover, comparing with the L. serrata reference sequences, we found only a single nucleotide change in our goat haplotype in 18s genetic region; while much nucleotide variations were observed in cox1 gene sequences. The results of the present study showed a high infection rate among goats and sheep in southeastern Iran. A better understanding of the disease could be achieved when the parasite species, their molecular characterization and the extent of infection in the area are determined. It is fundamental to select a comprehensive control program in order to take proper preventive and therapeutic measures against the infection.

Ubiquitous convalescent plasma: An artificial universal plasma for COVID-19 patients.

OBJECTIVES AND BACKGROUND: In December 2019, the first case of COVID-19 was reported in Wuhan, China. Its causative virus, is a novel strain of RNA viruses with high mortality rate. There is no definitive treatment, but among available approaches the use of recovered patients' plasma containing specific antibodies can enhance the immune response against coronavirus. However, the dearth of eligible donors and also ABO incompatibility in plasma transfusion, have limited this therapeutic method. Therefore, it is highly desirable to introduce a simple procedure that allows efficient reduction or even removal of natural ABO antibodies. Accordingly, we aimed to evaluate a RBC-mediated adsorption technique that reduces the titer of the mentioned antibodies in plasma. METHODS/MATERIALS: This experimental study was conducted in Kerman University of Medical Sciences, Kerman, Iran. The pre- and post-incubation antibody titers of 168 plasma samples were determined. For incubation, each plasma sample was exposed (60 min) to different percentages of RBCs at room temperature or 4 °C. RESULTS: The results evidenced that both the concentration of RBCs and temperature had significant decreasing effects on antibody titer (P < 0.001) and all concentrations significantly reduced titer. Compared to RT, 4 °C further reduced the antibody titer. Overall, the best incubation condition for reducing antibody titer in all blood groups was 4 °C and 2% RBCs concentration. CONCLUSION: The presented adsorption procedure is able to produce universal plasma (we call it Ubiquitous Convalescent Plasma) with a non-immunogenic level of ABO mismatch antibodies which can be used for COVID-19 patients with any type of blood group with desirable simplicity, feasibility, and efficacy.

Concomitant upregulation of nucleostemin and downregulation of Sox2 and Klf4 in gastric adenocarcinoma.

Nucleostemin (NS) is a nucleolar protein involved in stem cell (SC) self-renewal by controlling cell cycle progression. In addition to SCs, NS is also expressed in some highly proliferating cells including several adult stem cells and cancer cell lines. NS knock-down in different cell lines demonstrated its cell type-dependent function in arresting cell cycle in either G1 or G2/M phases. Here, we have evaluated the expression of NS and iPS genes in 36 gastric cancer and their matched marginal nontumor tissues by means of real-time polymerase chain reaction (RT-PCR). We have also examined a potential causative role of NS in gastric tumorigenesis by suppressing its expression in a gastric cancer cell line, AGS. Our data revealed that NS expression level is much higher in tumor tissues (p = 0.046), especially in high-grade ones (p < 0.001), whereas the expression of Klf4 and Sox2 is downregulated in tumor tissues compared to marginal nontumor samples (p < 0.001). Furthermore, NS suppression in the AGS cell line caused some morphological alterations, a cell cycle arrest at G1 phase, and an upregulation of iPS genes: Nanog, Sox2, and Klf4. Based on our results, NS overexpression seems to have a causative role in gastric tumorigenesis and/or progression, and it could be considered as a potential tumor marker for diagnosis, molecular classification, and molecular therapy of gastric adenocarcinoma.

Left Barrel Cortical Neurons Activity following Transplantation of Stem Cells into Right Lesioned-Barrel Cortex in Rats.

OBJECTIVE: Stem cells (SCs) can improve the functional defects of brain injury. Rodents use their whiskers to get tactile information from their surroundings. The aim of this study was to investigate whether the transplantation of SCs into the lesioned barrel cortex can help neuronal function in the contralateral cortex. MATERIALS AND METHODS: Sixteen male Wistar rats (200-230 g) were used in this experimental study. We induced a mechanical lesion in the right barrel cortex area of rats by removing this area by a 3 mm skin punch. Four groups containing one intact group of rats: group 1: control, and three lesion groups, group 2: lesion+un-differentiated dental pulp SCs (U-DPSCs), group 3: lesion+differentiated dental pulp SCs (D-DPSCs), and group 4: cell medium (vehicle) that were injected in the lesion area. Three weeks after transplantation of SCs or cell medium, the rats' responses of left barrel cortical neurons to controlled deflections of right whiskers were recorded by using the extracellular single-unit recordings technique. RESULTS: The results showed that the neural spontaneous activity and response magnitude of intact barrel cortex neurons in the lesion group decreased significantly (P<0.05) compared to the control group while ON and OFF responses were improved in the D-DPSCs (P<0.001) group compared to the vehicle group three weeks after transplantation. CONCLUSION: Transplantation of dental pulp mesenchymal SCs significantly improved the neural responses of the left barrel cortex that was depressed in the vehicle group.

Antileishmanial potentials of azacitidine and along with meglumine antimoniate on Leishmania major: In silico prediction and in vitro analysis.

This study aimed to investigate the in vitro and in silico antileishmanial activity of azacitidine (AZA) on Leishmania major promastigotes and amastigotes. The in silico method was used to evaluate the possibility of the interaction of AZA into the binding pocket of inducible nitric oxide synthase (iNOS), a leading defensive oxidative metabolite. Following that, in vitro anti-promastigote, and anti-amastigote activity of AZA was determined using an MTT assay and a macrophage model, respectively. Cytotoxic effects of AZA and meglumine antimoniate (MA) were also assessed by MTT assay on murine macrophages. All experiments were performed in triplicate. The results showed that AZA interacted with Ser133, Gln134, and Lys13 amino acids of iNOS, and the molecular docking score was obtained at -241.053 kcal/mol. AZA in combination with MA significantly (P<0.001) inhibited the growth rate of nonclinical promastigote (IC50 247.6±7.3 µM) and 8.5-fold higher of clinical intramacrophage amastigote stage (29.8±5.3 µM), compared to the untreated group. A significant upsurge of Th1 subsets and transcription genes and a meaningful decline in Th2 cytokines subclasses at the equivalent concentrations of AZA and MA was observed (P<0.001). The apoptosis effect of AZA along with MA was significantly induced on L. major in a dose-dependent manner (P<0.001). The present study demonstrated that AZA possesses antileishmanial activity in in vitro and in silico models. However, AZA combined with MA was more effective than AZA alone in inhibiting the growth rate of promastigotes and amastigotes of L. major. This study indicates that AZA in combination with MA demonstrated a potent antileishmanial mechanism, promoting immune response and enhancing an immunomodulatory role toward the Th1 pathway. This experimental study is a basic study for applying more knowledge about the mechanisms of AZA along with MA in animal models in the future.

Cytotoxicity effects of nanohybrid, bulk-fill, and ormocer composites on dental pulp stem cells and human gingival fibroblast cells.

Background: Despite significant improvements in the physical and esthetic properties of modern composite resins, there are still concerns about their biocompatibility. The aim of the current study was to evaluate the toxicity of X-tra fil, Grandio, and Admira Fusion composites on dental pulp stem cells (DPSCs) and human gingival fibroblast (HGF) cells. Materials and Methods: In this in vitro experimental study, 48 composite disks were made using Grandio, Admira Fusion (2 mm high and 4 mm in diameter), and X-tra fil (4 mm high and 4 mm in diameter) composites and cured for 40 s. The composite blocks were then crushed with a sterile mortar and dissolved in phosphate saline buffer solution. Tetrazolium salt (3-(4,5-dimethyl thiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT_, neutral red (NR) assay, flow cytometry, and quantitative real-time polymerase chain reaction (RT-PCR) tests (n = 5) were used to evaluate the toxicity of the composites on two cell types (HGF, DPSCs). Data were analyzed using one-way ANOVA test followed by Newman-Keuls test. Level of significance was set at P < 0.05. Results: According to the results of MTT test, only Grandio showed a significant cytotoxicity in DPSCs, but in HGF cells, Grandio and X-tra fil both showed a significant cytotoxicity. In NR test, Grandio and X-tra fil composites showed a significant cytotoxicity on both HGF and DPSC cells. RT-PCR test results on both DPSC and HGF cells indicated that bax gene expression in the Grandio composite was significant. In this test, the nonexpression of the bcl2 gene in DPSCs was significant in Grandio (100 and 200 µg/ml) and in X-tra fil (200 µg/ml). All of the tests performed in this study showed no significant toxicity of Admira fusion. Conclusion: Admira Fusion is suitable for oral cells in terms of biocompatibility and can be used as a suitable restorative material for deep restorations near the pulp or adjacent to the gums.

Comparing Periurethral Injection of Autologous Muscle-Derived Stem Cell and Fibroblasts with Mid-Urethral Sling Surgery in the Treatment of Female Stress Urinary Incontinence: A Randomized Clinical Trial.

Objective: In this study, we analyzed the therapeutic effect of periurethral injection of autologous muscle-derived stem cell versus mid-urethral sling surgery at a 1-year follow-up. Method: This randomized controlled clinical trial was conducted on 30 women with stress urinary incontinence (SUI) who had not responded to conservative treatments, after registering the participants and obtaining informed consent. Patients were divided into two groups of 15 each treated with periurethral injection of muscle-derived stem cells (MDSCs) and mid-urethral sling surgery, respectively. Follow-ups were done at 1, 3, 6, and 12 months after the treatment using the International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form (ICIQ-UISF) and Incontinence Quality of Life Questionnaire (I-QOL) questionnaires, clinical examination, cough test, and 1-hour pad test. The results were analyzed within the groups and then compared between the two groups. Moreover, both groups were compared in terms of postoperative complications. Results: At the 1-year follow-up, in the stem cell group, 10 patients (66.6%) experienced improvements after the periurethral injection of stem cells; half of these patients (33.3%) reported a full recovery. In the mid-urethral sling group, 13 patients (93.3%) experienced improvement, and 12 patients (80%) reported a full recovery. The analysis of ICIQ-UISF and I-QOL questionnaires indicated that the responses in both groups were significant, but the response in the stem cell group was significantly lower compared with the standard surgery group. No considerable complications were observed in the two groups. Conclusion: Although the periurethral injection of MDSCs considerably improves the symptoms with minimum complications in women with SUI, its therapeutic response is significantly lower compared with mid-urethral sling surgery.

The Efficacy of Kohl (Surma) and Erythromycin in Treatment of Blepharitis: An Open-Label Clinical Trial.

INTRODUCTION: Blepharitis is a common and chronic form of eyelid inflammation. Blepharitis treatment aims to decrease symptoms through antibacterial effects. One of the most common treatments of eyelid diseases in traditional medicine is using kohl. This clinical trial aimed to investigate its efficacy as a complementary treatment in staphylococcal blepharitis through an open-label clinical trial. MATERIALS AND METHODS: Thirty patients were randomized to receive kohl in one eye contralateral and erythromycin ointment in another eye for 90 days. At baseline and after 90 days of treatment, symptoms, clinical signs, and side effects of treatments were recorded. Statistical analysis was carried out using SPSS software, version 19. RESULTS: Despite randomization, there was a significant difference between the intervention and control eyes in the baseline mean clinical score (intervention eye: 9.86 (2.95) and control eye: 4.30 (2.81), P < 0.001). The degree of reduction of related signs and symptoms in the eyes treated with kohl was significantly higher than that in the control group: (5.2 vs. 2.20, P < 0.001) for symptoms and (7.40 vs. 2.46, P < 0.001) for clinical signs. Cohen's d statistic for mean difference of sign and symptom was 2.4 and 1.75, respectively, indicating a very strong effect. CONCLUSION: The present study results demonstrated a significant improvement in blepharitis-related signs and symptoms. The degree of improvement in the eyes treated with kohl was much higher than that in the control eyes.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus.

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Natural history of Echinococcus granulosus microcyst development in long term in vitro culture and molecular and morphological changes induced by insulin and BMP-4.

Introduction: Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus is a disease of worldwide public health and economic importance. The determinants and underlying cellular mechanisms of CE development and fate in intermediate hosts are largely unknown. Hormones and cytokines such as insulin and BMP-4 are the key players in the development, differentiation, and apoptosis. In this study, we evaluated the long term natural history of E. granulosus microcysts in an vitro setting and the molecular and morphological changes induced by the growth factors, insulin and BMP4 during the development of metacestode stage of E. granulosus. Methods: E. granulosus protoscoleces were cultivated and the parasite development was followed in the long term mono-phasic culture for 105 days and the morphometric, molecular and immunohistochemical changes were evaluated, including the microcysts number and size, microcysts development and deformation rates as well as the markers of calcification (Alizarin Red staining) and apoptosis (BAX, BCL2, Caspase-3, Caspase-8 and TNF-α expression) in the microcysts. Also the biological, histological and molecular consequences of insulin and BMP-4 treatment on the parasite development were evaluated. Results: Insulin and BMP-4 treatment of microcysts resulted in significant increase in microcyst formation, increased size, reduced apoptosis and deformation of the microcysts. Alizarin red staining of the microcysts treated with the insulin and BMP-4 confirmed that calcium deposition is significantly lower than the untreated microcysts. Also Alizarin Red staining and Immunohistochemistry of the microcysts indicates that calcium accumulation in deformed microcysts is higher than the normal ones on day 105. The microcysts began to wrinkle and the germinal layer was partially detached from the laminated layer on day 84. Conclusion: Results of the present study suggest that the degenerative changes in hydatid cysts can be slowed down by insulin and BMP-4, indicating that cellular factors and host hormones could contribute to the longevity of hydatid cysts. Significant evidences are provided suggesting that the microcysts cultivated in vitro can undergo calcification and apoptotic processes similar to what have been observed in the natural hydatid infection in the intermediate hosts.

Dynamic modeling of female neutering interventions for free-roaming dog population management in an urban setting of southeastern Iran.

Understanding dynamics of free-roaming dog (FRD) population is critical for planning and implementation of dog population management programs. FRD population size estimation as well as dynamic modeling of dog population under different female dog neutering interventions were investigated in order to determine the most appropriate animal birth control approach. We performed population size estimate of dogs using sight-resight surveys by photography in a randomly selected 25 blocks of the city and all the suburbs of greater Kerman area. Main demographic features were characterized and the dog density distribution was mapped. A dynamic model was developed to predict free-roaming dog population variations after 5 and 10 years. Different scenarios based on 10, 30, 50, 60 and 70% female dog sterilization were considered to predict the effects of animal birth control measures. Free roaming dog population was estimated at 6781 dogs (65.3% males) in Kerman and suburbs with several major population hotspots. Analysis of the dog locations within the city showed that the largest proportion of the dogs were observed in the vacant lots (46.2%). Modeling predictions indicated that, in the absence of management, the free-roaming dog population could increase from a baseline of 6781 to 13,665 dogs (2.02 fold increase) in 5 years and to 19,376 dogs in 10 years (2.86 fold increase). Using a population dynamics model, we simulated five neutering coverages to explore the impact of female neutering on free-roaming dog population size. The 5-year projections of the model have shown that 50% annual female dog sterilization significantly reduced free-roaming dog population by 0.44 comparing to the baseline population. Findings of the present study improve our knowledge on the nature and extent of dog population dynamics in Iran. Effective population control and selection of the most appropriate neutering interventions require a comprehensive knowledge of the characteristics and dynamics of FRD population.

Transplantation of rat dental pulp stem cells facilities post-lesion recovery in the somatosensory whisker cortex of male Wistar rats.

Damage to somatosensory "barrel" cortex reduces the rats' behavioral sensitivity in discrimination of tactile stimuli. Here, we examined how transplantation of stem cells into the lesioned barrel cortex can help in recovery of sensory capacities. We induced mechanical lesions in the right barrel cortex area of male rats. Three days after lesioning, rats received one of three transplantation types: un-differentiated dental pulp stem cells (U-DPSCs) or differentiated dental pulp stem cells (D-DPSCs), or cell medium (vehicle). A fourth group of rats were control without any Surgery. For 4 consecutive weeks, starting one week after transplantation, we evaluated the rats' preference to explore novel textures as a measure of sensory discrimination ability, also measured the expression of glial fibrillary acidic protein (GFAP), Olig 2, nestin, neuronal nuclei (NeuN), brain-derived neurotrophic factor (BDNF) and neuroligin1 by immunohistochemistry and western blotting. Unilateral mechanical lesion decreased the rats' preferential exploration of novel textures compared to the control group across the 4-week behavioral tests. Following stem cell therapy, the rats' performance significantly improved at week 2-4 compared to the vehicle group. Compared to the control group, there was a significant decrease in the expression of nestin, NeuN, Olig 2, BDNF, neuroligin1 and a significant increase in the expression of GFAP in the vehicle group. The expression of the neural markers was significantly higher in DPSCs compared with the vehicle group whereas GFAP level was lower in DPSCs compared to vehicle. We found that DPSCs therapy affected a range of neuronal markers in the barrel cortex post lesion, and improved the rats' recovery for sensory discrimination.

Cord blood serum harvesting by hydroxyethyl starch: a fetal bovine serum alternative in expansion of umbilical cord-derived mesenchymal stem cells.

As a widely used cell culture supplement, fetal bovine serum (FBS) harbor high content of growth, proliferation, and adhesion factors. However, high cost, bio-safety, possible xenogeneic agent transmission, finite accessible, and ethical controversy are major obstacles that discourage the use of this additive. Accordingly, novel alternatives have been proposed with various pros and cons. Still, caution should be taken in choosing suitable substitute given that the alteration in the main aspects of cultured cells can be biased the consequences of clinical applications. Herein, the authors evaluated the impact of cord blood serum harvesting by hydroxyethyl starch (CBS-HES), as an enriched source of growth factors, on the basic mesenchymal stem cells (MSCs) characteristics. In the present experiment, umbilical cord-derived MSCs were isolated and continuously nourished with Dulbecco's Modified Eagle Medium containing either 10, 15, and 20% CBS-HES or FBSs to compare their morphology, immunophenotype, growth and proliferation rate, death rate, cell cycle, and gene expression profiles. Although all enriched media supported the expansion of MSCs with comparable morphology, cell surface markers, death rate, c-MYC and p16 expression, and growth rate, CBS-HES treated cells significantly (P < 0.05) expressed more hTERT gene in a concentration-dependent manner. Yet no significant shift was observed in the cell cycle of cultured cells using the same concentrations of additives, a finding which further confirmed by Ki-67 immunostaining. CBS-HES as an available and affordable additive, seems to be an optimal, relatively safe, and promising FBS alternative for cultivation, propagation, and subsequent clinical applications of MSCs.

A fibrinous and allogeneic fibroblast-enriched membrane as a biocompatible material can improve diabetic wound healing.

The application of conventional approaches to diabetic wound regeneration has some limitations. Thus, skin substitutes could be a new therapeutic possibility. In this regard, fibrin scaffolds are promising materials due to their desirable characteristics. Since defective fibroblasts caused by diabetes can disrupt regeneration, it seems that the use of living cells can improve the healing process. Thus, based on this fact, a cellular fibrin membrane was used to evaluate the diabetic wound healing in rats. The fibrin membrane was fabricated using fresh frozen plasma on which isolated fibroblasts were cultured. The wound model was created on 36 diabetic rats that were randomly divided into three groups: control, membrane, and cellular fibrin membrane (CM). Wound photogramography and immuno-histopathological staining were performed during consecutive days after treatment. Macroscopic evaluation of the wounds indicated a noteworthy enhancement of wound closure in the CM group. In the CM group, the re-epithelialization rate on day 7, 10 (p < 0.001), and 14 (p < 0.05), the fibroblast percentage on day 3 (p < 0.01) and 7 (p < 0.05) and the collagenization in all days were significantly higher than those of other groups (p < 0.001). The fibroblast number in the CM group on day 10 was significantly (p < 0.01) lower than that in the other groups. Contrary to the neutrophil and angiogenesis percentages that had no significant difference among the groups at different points of time (p > 0.05), the macrophage percentage on day 7 (P < 0.01), 10, and 14 (p < 0.05) was significantly lower in the CM group as compared to that in other groups. Overall, it seems that the use of a fibroblast-loaded fibrin membrane is an attractive strategy to promote diabetic wound healing.

Use of Some Additives for Improving Mesenchymal Stem Cell Isolation Outcomes in Non-Mobilized Peripheral Blood.

BACKGROUND: The mesenchymal stem cells (MSCs) of peripheral blood (PB) have been recognized as a promising source for allogeneic cell therapy. The objective of the present study was to isolate and characterize MSCs derived from non-mobilized PB, and evaluate their differentiation potential. METHODS: The buffy coat mononuclear fractions of the PB were concentrated using the Ficoll-Paque density gradient centrifugation and were grown on primary and secondary culture media, respectively. The isolated cells were characterized using a multidisciplinary approach which was based on morphology, immunophenotyping, gene expression, and multipotentiality. Flow cytometry and Reverse transcription polymerase chain reaction (RT-PCR) were used to identify the expression of different MSC markers. Finally, after culturing in osteogenic and adipogenic induction media, the isolated cells were stained by Alizarin red and Oil-Red O. RESULTS: In spite of absence of any bone marrow stimulating factor, the isolation approach in this study yielded a rather homogeneous and spindle-shaped mononuclear cell population (the yield of passage 0 was 0.65 ± 0.15) that stained positive for CD90, CD105, and CD73, and were negative for CD45 and CD34. These cells have high proliferative capacity (confirmed by the expression of Oct-4, Nucleostemin, and Nanog genes) and were able to differentiate into lineage-specific committed cells, when exposed to the appropriate medium. CONCLUSION: Overall, it can be concluded that conventional, labour-intensive and time-consuming approaches are not necessary in isolating MSCs from PB. This relatively accessible and minimally invasive source, PB, represents a good alternative reservoir of homogeneous MSCs that could open a new era for practical exploitation in regenerative medicine.

Application and Assessment of Allogeneic Fibroblasts for Cell Therapy.

BACKGROUND AND OBJECTIVE: In recent years, due to increasing number of patients with non-healing skin ulcers, skin substitutes have been used. Skin substitutes contain living cells causing faster and more effective wound healing. Therefore, research on the use of autologous and allogeneic cells such as fibroblasts in skin substitutes has attracted attentions. However, there are discrepancies in the immune responses to allogeneic fibroblasts. Therefore, we aimed to review the immune responses to allogeneic fibroblasts. METHODS: Donor fibroblasts were isolated from the skin of three rats. Nine recipient rats which were subcutaneously injected with three different regimens, were divided into three groups: Group 1; phosphate buffered saline (PBS) without cells (control), group 2: allogeneic fibroblasts of one animal source suspended in phosphate buffered saline, and group 3; phosphate buffered saline containing mixed allogeneic fibroblasts of three animal sources. The skin samples were biopsied at 1, 3 and 7 days after injection and studied histopathologically. RESULTS AND CONCLUSION: No signs of redness and edema were observed in the injection sites. In pathology examination, changes such as vasculitis, eosinophils and lymphocytes accumulation around fibroblasts, fibroblast apoptosis and transplant rejection at the injection site were not observed in either group.Subcutaneous injection of allogeneic fibroblasts in rats can be introduced as a promising approach for wound healing as they do not stimulate the immune system.

Peripheral Blood-Derived Mesenchymal Stem Cells: Growth Factor-Free Isolation, Molecular Characterization and Differentiation.

BACKGROUND AND OBJECTIVES: The mesenchymal stem cells derived from peripheral blood (PB) have been recognized as a promising source for allogeneic cell therapy. The aim of this study was to investigate the isolation, growth and differentiation ability of peripheral blood-isolated mesenchymal stem cells. METHODS: The mononuclear cells were purified from fresh peripheral blood using density gradient centrifugation then cultured in a suitable medium, expanded and characterized. In the following, these cells were cultured in specific adipogenic and osteogenic differentiation media. RESULTS AND CONCLUSION: In spite of the absence of any stimulating factor, the cells adhered to the flasks and developed a rather homogeneous, spindle-shaped morphology after consecutive passages. The cells were confirmed to have mesenchymal phenotype by expression of specific markers (CD90, CD105, and CD73) and absence of CD45 marker, which is specific for hematopoietic stem cells. They could differentiate into lineage-specific committed cells (osteoblasts and adipocytes).According to the findings, the conventional, labour-intensive and time-consuming approaches are not necessary to obtain an optimal number of cells from peripheral blood. This relatively accessible and minimally invasive source of stem cells may open a new era for practical exploitation in regenerative medicine.

Isolation and evaluation of dental pulp stem cells from teeth with advanced periodontal disease.

INTRODUCTION: Successful isolation of mesenchymal stem cells from waste tissues might be extremely promising for developing stem cell-based therapies. This study aimed to explore whether cells retrieved from teeth extracted due to advanced periodontal disease present mesenchymal stem cell-like properties. METHODS: Pulp cells were isolated from 15 intact molars and 15 teeth with advanced periodontal disease. Cell proliferation and markers of mesenchymal stem cells were evaluated. RESULTS: Based on the RT-PCR and agarose gel electrophoresis, nucleostemin, Oct-4 and jmj2c, but not Nanog, were expressed in undifferentiated mesenchymal stem cells of both groups. Interestingly, diseased pulp exhibited higher gene expressions although it was not statistically significant. The average percentage of BrdU positive cells in the diseased group (84.4%, n = 5) was significantly higher than that of the control group (65.4%, n = 5) (t-test, P = 0.001). CONCLUSION: Our results indicate the successful isolation of mesenchymal stem cells from the pulp tissue of hopeless periodontally involved teeth.

A modified efficient method for dental pulp stem cell isolation.

BACKGROUND: Dental pulp stem cells can be used in regenerative endodontic therapy. The aim of this study was to introduce an efficient method for dental pulp stem cells isolation. MATERIALS AND METHODS: In this in-vitro study, 60 extracted human third molars were split and pulp tissue was extracted. Dental pulp stem cells were isolated by the following three different methods: (1) digestion of pulp by collagenase/dispase enzyme and culture of the released cells; (2) outgrowth of the cells by culture of undigested pulp pieces; (3) digestion of pulp tissue pieces and fixing them. The cells were cultured in minimum essential medium alpha modification (αMEM) medium supplemented with 20% fetal bovine serum(FBS) in humid 37°C incubator with 5% CO 2. The markers of stem cells were studied by reverse transcriptase polymerase chain reaction (PCR). The student t-test was used for comparing the means of independent groups. P <0.05 was considered as significant. RESULTS: The results indicated that by the first method a few cell colonies with homogenous morphology were detectable after 4 days, while in the outgrowth method more time was needed (10-12 days) to allow sufficient numbers of heterogeneous phenotype stem cells to migrate out of tissue. Interestingly, with the improved third method, we obtained stem cells successfully with about 60% efficiency after 2 days. The results of RT-PCR suggested the expression of Nanog, Oct-4, and Nucleostemin markers in the isolated cells from dental pulps. CONCLUSION: This study proposes a new method with high efficacy to obtain dental pulp stem cells in a short time.