Cell Journal (Yakhteh)
Volume:16 Issue: 1, Spring 2014

We introduce an RGD (Arg-Gly-Asp)-containing peptide of collagen IV origin that possesses potent cell adhesion and proliferation properties. In this experimental study, the peptide was immobilized on an electrospun nanofibrous polycaprolactone/gelatin (PCL/Gel) hybrid scaffold by a chemical bonding approach to improve cell adhesion properties of the scaffold. An iodine-modified phenylalanine was introduced in the peptide to track the immobilization process. Native and modified scaffolds were characterized with scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). We studied the osteogenic and adipogenic differentiation potential of human bone marrow-derived mesenchymal stem cells (hBMSCs). In addition, cell adhesion and proliferation behaviors of hBMSCs on native and peptide modified scaffolds were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4'',6-diamidino-2-phenylindole (DAPI) staining, and the results compared with tissue culture plate, as the control. FTIR results showed that the peptide successfully immobilized on the scaffold. MTT assay and DAPI staining results indicated that peptide immobilization had a dramatic effect on cell adhesion and proliferation. This peptide modified nanofibrous scaffold can be a promising biomaterial for tissue engineering and regenerative medicine with the use of hBMSCs.

Gelatinases are a large group of proteolytic enzymes that belong to the matrix metalloproteinases (MMPs). MMPs are a broad family of peptidases, which proteolyse the extracellular matrix and have an important role in inflammation. Verapamil is a calcium channel blocker extensively used in the treatment of numerous cardiovascular diseases such as arrhythmia and hypertension. The anti-tumor and anti-inflammatory effects of verapamil have also been shown. In this study, the effect of verapamil on gelatinase activity in human peripheral blood mononuclear cells (PBMCs) has been assessed in vitro. In this experimental study, PBMCs from healthy adult volunteers were isolated by ficoll-hypaque-gradient centrifugation. The cells were then cultured in complete RPMI-1640 medium and after that incubated with different concentrations of verapamil (0–200 μM) in the presence or absence of phytoheamagglutinin (PHA) (10 μg/ml) for 48 hours. The gelatinase A (MMP-2)/gelatinase B (MMP-9) activity in cell-conditioned media was then evaluated by gelatin zymography. Statistical comparisons between groups were made by analysis of variance (ANOVA). Verapamil significantly decreased the MMP-2/MMP-9 activity in human PBMCs after 48 hours incubation time compared with untreated control cells. The association was dose-dependent. In this study verapamil exhibited a dose-dependent inhibitory effect on gelatinase A and gelatinase B activity in human PBMCs. It seems that the anti-inflammatory properties of verapamil may be in part due to its inhibitory effects on gelatinase activity. Regarding the beneficial effects of MMPs- inhibitors in the treatment of some cardiovascular diseases, the positive effect of verapamil on such diseases may be in part due to its anti-MMP activity. Verapamil with its inhibitory effects on gelatinases activity may be a useful MMP-inhibitor. Given the beneficial effect of MMP-inhibitors in some cancerous, inflammatory and autoimmune disorders, it seems likely that verapamil could also be used to treat these diseases.

Colorectal cancer (CRC) is one of the most common and aggressive cancers worldwide. The majority of CRC cases are sporadic that caused by somatic mutations. The Adenomatous Polyposis Coli (APC; OMIM 611731) is a tumor suppressor gene of Wnt pathway and is frequently mutated in CRC cases. This study was designed to investigate the spectrum of APC gene mutations in Iranian patients with sporadic colorectal cancer. In this descriptive study, Tumor and normal tissue samples were obtained from thirty randomly selected and unrelated sporadic CRC patients. We examined the hotspot region of the APC gene in all patients. Our mutation detection method was direct DNA sequencing. We found a total of 8 different APC mutations, including two nonsense mutations (c.4099C>T and c.4348C>T), two missense mutations (c.3236C>G and c.3527C>T) and four frame shift mutations (c.2804dupA, c.4317delT, c.4464_4471delATTACATT and c.4468_4469dupCA). The c.3236C>G and c.4468_4469dupCA are novel mutations. The overall frequency of APC mutation was 26.7% (8 of 30 patients). This mutation rate is lower in comparison with previous studies from other countries. The findings of present study demonstrate a different APC mutation spectrum in CRC patients of Iranian origin compared with other populations.

Olive oil and olive leaf extract are used for treatment of skin diseases and wounds in Iran. The main component of olive leaf extract is Oleuropein. This research is focused on the effects of Oleuropein on skin wound healing in aged male Balb/c mice. In this experimental study, Oleuropein was provided by Razi Herbal Medicine Institute, Lorestan, Iran. Twenty four male Balb/c mice, 16 months of age, were divided equally into control and experimental groups. Under ether anesthesia, the hairs on the back of neck of all groups were shaved and a 1 cm long full-thickness incision was made. The incision was then left un-sutured. The experimental group received intradermal injections with a daily single dose of 50 mg/kg Oleuropein for a total period of 7 days. The control group received only distilled water. On days 3 and 7 after making the incision and injections, mice were sacrificed, and the skin around incision area was dissected and stained by hematoxylin and eosin (H&E) and Van Gieson’s methods for tissue analysis. In addition, western blot analysis was carried out to evaluate the level of vascular endothelial growth factor (VEGF) protein expression. The statistical analysis was performed using SPSS (SPSS Inc., Chicago, USA). The t test was applied to assess the significance of changes between control and experimental groups. Oleuropein not only reduced cell infiltration in the wound site on days 3 and 7 post incision, but also a significant increase in collagen fiber deposition and more advanced re- epithelialization were observed (p<0.05) in the experimental group as compared to the control group. The difference of hair follicles was not significant between the two groups at the same period of time. Furthermore, western blot analysis showed an increased in VEGF protein level from samples collected on days 3 and 7 post-incision of experimental group as compared to the control group (p<0.05). These results suggest that Oleuropein accelerates skin wound healing in aged male Balb/c mice. These findings can be useful for clinical application of Oleuropein in expediting wound healing after surgery.

Our research attempted to show that mouse dental pulp stem cells (DPSCs) with characters such as accessibility, propagation and higher proliferation rate can provide an improved approach for generate bone tissues. With the aim of finding and comparing the differentiation ability of mesenchymal stem cells derived from DPSCs into osteoblast and osteoclast cells; morphological, molecular and biochemical analyses were conducted. In this experimental study, osteoblast and osteoclast differentiation was induced by specific differentiation medium. In order to induce osteoblast differentiation, 50 μg mL-1 ascorbic acid and 10 mM β-glycerophosphate as growth factors were added to the complete medium consisting alpha-modified Eagle’s medium (α-MEM), 15% fetal bovine serum (FBS) and penicillin/streptomycin, while in order to induce the osteoclast differentiation, 10 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL) and 5 ng/mL macrophage-colony stimulating factor (M-CSF) were added to complete medium. Statistical comparison between the osteoblast and osteoclast differentiated groups and control were carried out using t test. Proliferation activity of cells was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Statistical results demonstrated significant difference (p<0.05) between the control and osteoblastic induction group, whereas osteoclast cells maintained its proliferation rate (p>0.05). Morphological characterization of osteoblast and osteoclast was evaluated using von Kossa staining and May-Grunwald-Giemsa technique, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) molecular analysis demonstrated that mouse DPSCs expressed Cd146 and Cd166 markers, but did not express Cd31, indicating that these cells belong to mesenchymal stem cells. Osteoblast cells with positive osteopontin (Opn) marker were found after 21 days, whereas this marker was negative for DPSCs. CatK, as an osteoclast marker, was negative in both osteoclast differentiation medium and control group. Biochemical analyses in osteoblast differentiated groups showed alkaline phosphatase (ALP) activity significantly increased on day 21 as compared to control (p<0.05). In osteoclast differentiated groups, tartrate-resistant acid phosphatase (TRAP) activity representing osteoclast biomarker didn’t show statistically significant as compared to control (p>0.05). DPSCs have the ability to differentiate into osteoblast, but not into osteoclast cells.

Finding cell sources for cartilage tissue engineering is a critical procedure. The purpose of the present experimental study was to test the in vitro efficacy of the beta-tricalcium phosphate-alginate-gelatin (BTAG) scaffold to induce chondrogenic differentiation of human umbilical cord blood-derived unrestricted somatic stem cells (USSCs). In this experimental study, USSCs were encapsulated in BTAG scaffold and cultured for 3 weeks in chondrogenic medium as chondrogenic group and in Dulbecco’s Modified Eagle’s Medium (DMEM) as control group. Chondrogenic differentiation was evaluated by histology, immunofluorescence and RNA analyses for the expression of cartilage extracellular matrix components. The obtain data were analyzed using SPSS version 15. Histological and immunohistochemical staining revealed that collagen II was markedly expressed in the extracellular matrix of the seeded cells on scaffold in presence of chondrogenic media after 21 days. Reverse transcription-polymerase chain reaction (RT-PCR) showed a significant increase in expression levels of genes encoded the cartilage-specific markers, aggrecan, type I and II collagen, and bone morphogenetic protein (BMP)-6 in chondrogenic group. This study demonstrates that BTAG can be considered as a suitable scaffold for encapsulation and chondrogenesis of USSCs.

Biomaterial technology, when combined with emerging human induced pluripotent stem cell (hiPSC) technology, provides a promising strategy for patient-specific tissue engineering. In this study, we have evaluated the physical effects of collagen scaffolds fabricated at various freezing temperatures on the behavior of hiPSC-derived neural progenitors (hiPSC-NPs). In addition, the coating of scaffolds using different concentrations of laminin was examined on the cells. Initially, in this experimental study, the collagen scaffolds fabricated from different collagen concentrations and freezing temperatures were characterized by determining the pore size, porosity, swelling ratio, and mechanical properties. Effects of cross-linking on free amine groups, volume shrinkage and mass retention was also assessed. Then, hiPSC-NPs were seeded onto the most stable three-dimensional collagen scaffolds and we evaluated the effect of pore structure. Additionally, the different concentrations of laminin coating of the scaffolds on hiPSC-NPs behavior were assessed. Scanning electron micrographs of the scaffolds showed a pore diameter in the range of 23-232 μm for the scaffolds prepared with different fabrication parameters. Also porosity of all scaffolds was >98% with more than 94% swelling ratio. hiPSC-NPs were subsequently seeded onto the scaffolds that were made by different freezing temperatures in order to assess for physical effects of the scaffolds. We observed similar proliferation, but more cell infiltration in scaffolds prepared at lower freezing temperatures. The laminin coating of the scaffolds improved NPs proliferation and infiltration in a dose-dependent manner. Immunofluorescence staining and scanning electron microscopy confirmed the compatibility of undifferentiated and differentiated hiPSC-NPs on these scaffolds. The results have suggested that the pore structure and laminin coating of collagen scaffolds significantly impact cell behavior. These biocompatible three-dimensional laminin-coated collagen scaffolds are good candidates for future hiPSC-NPs biomedical nerve tissue engineering applications.

In vitro production of a definitive endoderm (DE) is an important issue in stem cell-related differentiation studies and it can assist with the production of more efficient endoderm derivatives for therapeutic applications. Despite tremendous progress in DE differentiation of human embryonic stem cells (hESCs), researchers have yet to discover universal, efficient and cost-effective protocols. In this experimental study, we have treated hESCs with 200 nM of Stauprimide (Spd) for one day followed by activin A (50 ng/ml; A50) for the next three days (Spd-A50). In the positive control group, hESCs were treated with Wnt3a (25 ng/ml) and activin A (100 ng/ml) for the first day followed by activin A for the next three days (100 ng/ml; W/A100-A100). Gene expression analysis showed up regulation of DE-specific marker genes (SOX17, FOXA2 and CXCR4) comparable to that observed in the positive control group. Expression of the other lineage specific markers did not significantly change (p<0.05). We also obtained the same gene expression results using another hESC line. The use of higher concentrations of Spd (400 and 800 nM) in the Spd-A50 protocol caused an increase in the expression SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration of activin A (25 ng/ml) was not able to up regulate the DE-specific marker genes. Then, A50 was replaced by inducers of definitive endoderm; IDE1/2 (IDE1 and IDE2), two previously reported small molecule (SM) inducers of DE, in our protocol (Spd-IDE1/2). This replacement resulted in the up regulation of visceral endoderm (VE) marker (SOX7) but not DE-specific markers. Therefore, while the Spd-A50 protocol led to DE production, we have shown that IDE1/2 could not fully replace activin A in DE induction of hESCs. These findings can assist with the design of more efficient chemically-defined protocols for DE induction of hESCs and lead to a better understanding of the different signaling networks that are involved in DE differentiation of hESCs.

Introduction of new approaches for the treatment of human immunodeficiency virus (HIV) infection such as anti-retroviral medicines has resulted in an increase in the life expectancy of HIV patient. Evaluating the dental health status as a part of their general health care is needed in order to improve the quality of life in these patients. The aim of this study was to compare the root and crown caries rate in HIV patients receiving highly active antiretroviral therapy (HAART) with that rate in HIV patients without treatment option. This cross sectional study consisting of 100 individuals of both genders with human immunodeficiency virus were divided into two groups: i. group 1 (treatment group) including 50 patients with acquired immunodeficiency syndrome (AIDS) receiving HAART and ii. group 2 (control group) including 50 HIV infected patients not receiving HAART. Dental examinations were done by a dentist under suitable light using periodontal probe. For each participant, numbers of decay (D), missed (M), filled (F), Decayed missed and filled teeth (DMFT), decay surface (Ds), missed surface (Ms), filled surface (Fs), Decayed missed and filled surfaces (DMFS), and tooth and root caries were recorded. Data were analyzed using Chi-square test and independent t test using SPSS 13.0, while p-value of <0.05 was considered statistically significant in all analysis. The mean and standard deviation (SD) of decayed, missed and filled teeth of those who were on highly active antiretroviral therapy was 6.86 ± 3.57, 6.39 ± 6.06 and 1.89 ± 1.93, respectively. There was no significant difference between these values regarding to the treatment of patients. The mean and standard deviation of DMFT, DMFS and the number of decayed root surfaces were 15.14 ± 6.09, 56.79 ± 28.56, and 4.96 ± 2.89 in patients treated by anti-retroviral medicine which were not significantly different compared to those without this treatment. According to the results of the present study, highly active antiretroviral therapy could not be considered as a single factor for dental caries prevalence in HIV-infected patients. However, more research is recommended to evaluate the cariogenic potential of these medicines.

Spermatogonial stem cells (SSCs) are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional (3D) culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting (MACS) using GDNF family receptor alpha-1 (Gfrα-1) antibody. Two experimental designs were investigated. 1. Gfrα-1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfrα-1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 (SCP3)-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 (TTF1) as post meiotic markers. For statistical analysis student t test was performed. Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro.

Fibrodysplasia Ossificans Progressiva (FOP, MIM 135100) is a rare genetic disease that is often inherited sporadically in an autosomal dominant pattern. The disease manifests in early life with malformed great toes and, its episodic and progressive bone formation in skeletal muscle after trauma is led to extra-articular ankylosis. In this study, a 17 year-old affected girl born to a father with chemical injury due to exposure to Mustard gas during the Iran-Iraq war, and her first degree relatives were examined to find the genetic cause of the disease. The mutation c.617G>A in the Activin A receptor, type I (ACVR1) gene was found in all previously reported patients with FOP. Therefore, peripheral blood samples were taken from the patient and her first-degree relatives. DNA was extracted and PCR amplification for ACVR1 was performed. The sequencing of ACVR1 showed the existence of the heterozygous c.617G>A mutation in the patient and the lack of it in her relatives. Normal result of genetic evaluation in relatives of the patient, ruled out the possibility of the mutation being inherited from parents. Therefore, the mutation causing disease in the child, whether is a new mutation with no relation to the father’s exposure to chemical gas, or in case of somatic mutation due to exposure to chemical gas, the mutant cells were created in father’s germ cells and were not detectable in his blood sample.

Small cell carcinoma of the urinary bladder (SCCUB) is an extremely rare bladder malignancy characterized by an aggressive clinical behavior. So, it is important to diagnose this high grade disease by urinary cytology. We report a case of SCCUB in an old man with chronic lymphocytic leukemia (CLL) in remission, while bladder tumor was diagnosed by cytology. With this article, we aimed to review and to update the literature concerning this tumor.