Cell Journal (Yakhteh)
Volume:13 Issue: 4, Winter 2012

One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importance of mycoplasma tests in cell culture; 4. different methods to identify mycoplasma contamination; 5. the consequences of mycoplasma contamination in cell culture and 6. available methods to eliminate mycoplasma contamination. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture.

Several studies have shown that, although transplantation of neural stem cells into the contusion model of spinal cord injury (SCI) promotes locomotor function and improves functional recovery, it induces a painful response, Allodynia. Different studies indicate that bone marrow stromal cells (BMSCs) and Schwann cells (SCs) can improve locomotor recovery when transplanted into the injured rat spinal cord. Since these cells are commonly used in cell therapy, we investigated whether co-transplantation of these cells leads to the development of Allodynia. In this experimental research, the contusion model of SCI was induced by laminectomy at the T8-T9 level of the spinal cord in adult female wistar rats (n=40) weighting (250-300g) using the New York University Device. BMSCs and SCs were cultured and prelabeled with 5-bromo-2-deoxyuridine (BrdU) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) respectively. The rats were divided into five groups of 8 including: a control group (laminectomy only), three experimental groups (BMSC, SC and Co-transplant) and a sham group. The experimental groups received BMSCs, SCs, and BMSCs and SCs respectively by intraspinal injection 7 days after injury and the sham group received serum only. Locomotion was assessed using Basso, Beattie and Bresnahan (BBB) test and Allodynia by the withdrawal threshold test using Von Frey Filaments at 1, 7, 14, 21, 28, 35, 42, 49 and 56 days after SCI. The statistical comparisons between groups were carried out by using repeated measures analysis of variances (ANOVA). Significant differences were observed in BBB scores in the Co- transplant group compared to the BMSC and SC groups (p< 0.05). There were also significant differences in the withdrawal threshold means between animals in the sham group and the BMSC, SC and the Co-transplant groups (p<0.05).BBB scores and withdrawal threshold means showed that co-transplation improved functioning but greater Allodynia compared to the other experimental groups. The present study has shown that, although transplantation of BMSCs, SCs and a combination of these cells into the injured rat spinal cord can improve functional recovery, it leads to the development of mechanical Allodynia. This finding indicates that strategies to reduce Allodynia in cell transplantation studies are required.

Evaluation of the effect of Propolis as a bioactive material on quality of dentin and presence of dental pulp stem cells. For conducting this experimental split-mouth study,a total of 48 maxillary and mandibular incisors of male guinea pigs were randomly divided into an experimental Propolis group and a control calcium hydroxide group. Cutting the crowns and using Propolis or calcium hydroxide to cap the pulp, all of the cavities were sealed. Sections of the teeth were obtained after sacrificing 4 guinea pigs from each group on the 10th, 15th and 30th day. After they had been stained by hematoxylin and eosin (H&E), specimens underwent a histological evaluation under a light microscope for identification of the presence of odontoblast-like cells, pulp vitality, congestion, inflammation of the pulp and the presence of remnants of the material used. The immunohistochemistry (IHC) method using CD29 and CD146 was performed to evaluate the presence of stem cells and the results were statistically evaluated by Kruskal-Wallis, Chi Square and Fisher tests. In H&E stained specimens, there was no difference between the two groups in the presence of odontoblast-like cells, pulp vitality, congestion, inflammation of the pulp and the presence of remnants of used material(p>0.05). There was a significant difference between the quality of regenerative dentin on the 15th and 30th days (p<0.05): all of the Propolis cases presented tubular dentin while 14% of the calcium hydroxide cases produced porous dentin. There was no significant difference between Propolis and calcium hydroxide in stimulation of dental pulp stem cells (DPSCs). This study which is the first one that documented the stimulation of stem cells by Propolis, provides evidence that this material has advantages over calcium hydroxide as a capping agent in vital pulp therapy. In addition to producing no pulpal inflammation, infection or necrosis this material induces the production of high quality tubular dentin.

Age-related changes occur in many different systems of the body. Many elderly people show dysphagia and dysphonia. This research was conducted to evaluate quantitatively the morphometrical changes of the hypoglossal nerve resulting from the aging process in young and aged rats. Through an experimental study ten male wistar rats (4 months: 5 rats, 24 months: 5 rats) were selected randomly from a colony of wistars in the UWC. After a fixation process and preparation of samples of the cervical portion of the hypoglossal nerve of these rats, light and electron microscopic imaging were performed. These images were evaluated according to the numbers and size of myelinated nerve fibers, nucleoli of Schwann cells, myelin sheath thickness, axon diameter, and g ratio. All data were analyzed by Mann-Whitney, a non-parametric statistical test. In light microscope, numbers of myelinated nerve fibers, the mean entire nerve perimeters, the mean entire nerve areas and the mean entire nerve diameters in young and aged rats’ were not significantly different between the two groups. In electron microscope, numbers of myelinated axons, numbers of Schwann cell nucleoli and the mean g ratios of myelinated axon to Schwann cell in young and aged rats were not significantly different. The myelinated fiber diameters, the myelin sheath thicknesses, myelinated axon diameters and the mean g ratio of axon diameter to myelinated fiber diameter in young and aged fibers were significantly different The mean g ratio of myelinated nerve fibers of peripheral nerves stabilizes at the level of 0.6 after maturation and persists without major change during adulthood. This ratio of axon diameter to fiber diameter (0.6) is optimum for normal conduction velocity of neural impulses. Our study indicated that the g ratio of myelinated nerve fiber of the hypoglossal nerve decreased prominently in aged rats and can be a cause of impairment in nerve function in old age. Thus, prospective studies concerning electrophysiological and conductive properties of the peripheral nerve could be useful to clarify further the effects of aging on peripheral nerves.

A reduction in new human immunodeficiency virus(HIV) cases is one of the ten areas prioritized by the United Nations Program on HIV. However, recent official reports confirm the HIV rate is increasing and predicted a huge incidence in the near future in Iran, despite the preventative program by Iran’s Health Ministry. In this descriptive study, we evaluate the frequency of HIV positive cases among referral patients to a private clinic laboratory for its diagnosis in addition to specimens from other laboratories. An epidemiological analysis is also performed. In this descriptive study, the total number of patients was 138 cases that referred for the diagnosis of HIV to the private Laboratory. Of these, 93 males (67.4%) and 45 females (32.6%) voluntarily requested to be examined for specific increases in specific antibody titer, western blot assays and RNA quantitation polymerase chain reaction. We collected two separate tubes of whole blood, one for reverse transcriptasepolymerase chain reaction analysis and the second one for the remaining two tests. Those patients who were antibody positive by western blot and/or reverse transcriptase-polymerase chain reaction(RT-PCR) analyses were considered as HIV positive cases. There were 18.84% confirmed HIVcases (17.39% males; 1.45% females). Analysis of the results confirmed that the ratio of male to female patients in the infected group was not comparable to those in the suspect group. The majority of HIV positive cases were either infected by their partner via sexual intercourse (84.61%) or needle sticks (11.53%) among the drug addicted group. The infection routes of the remainder were unknown. Analysis of the data revealed a higher frequency of HIVin males than females among the tested group. There was a shift in to unsafe sexual intercourse as seen in the present study. The higher rate of infected male patients shows a shift in transmission route to unsafe intercourse. Therefore, it is necessary to design new supportive programs by actively identifying and contacting at-risk groups, particularly infected females who are uninterested in being and monitored.

It has been reported that rat bone marrow stromal cells (BMSCs) can be spontaneously differentiated into neural-like cells without any supplemental growth factors and/or chemical treatment after long-term culture.This study aims to determineWhether, growth factors secreted by MSCs could induce self-differentiation into neural-like cells in a long-term culture. This study consisted of two groups: i. rat BMSCs (passage 5) were cultured in alfa- minimal essential medium (α-MEM) and 10% fetal bovine serum (FBS) without the addition of inducer and exchanging medium for three weeks, as the experimental group and ii.rat BMSCs (passage 5) as the control group. Each group was analysed by reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the expressions of neurotrophic factors and neural marker genes. Statistical analyses were carried out using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison with SPSS software (version 16). P< 0.05 was considered statistically significant. The experimental group (fifth passage of BMSCs) obtained from adult rats spontaneously differentiated into neural precursor cells after long-term culture. Cultured cells expressed tyrosine hydroxylase (TH), Nurr1 and nestin genes. Furthermore, some growing cells in suspension became neurosphere-like. Self-differentiated rat MSCs (SDrMSCs) expressed significantly higher levels of NGF (0.96 ± 0.16), nestin (0.63 ± 0.08), and Nurr1 (0.80 ± 0.10) genes (p<0.05). In this study, we reported that rMSCs in long-term culture underwent spontaneous transformation to neural precursors without the supplement of growth factors and specific chemicals. Cells expressed neural markers such as: TH, Nurr1, and nestin genes.

microRNAs (miRNAs) are a new class of non-coding RNAs involved in regulating various biological processes including proliferation, differentiation, and apoptosis, among others. Alterations in miRNA expression are reported in several human cancers, which suggests their potential roles in tumor initiation and progression. Members of the miR-302 cluster are highly expressed in embryonic stem cells (ESC), where they regulate cell self-renewal and pluripotency. Based on the cancer stem cell (CSC) hypothesis, mis-expression of such genes might contribute to tumorigenicity. This study aims to find a potential link between the expression level of human/homo sapiens miR-302b (has-miR- 302b) and tumor/grade state of gastric tissues. A matched based case-control study was conducted that included tumor and matched marginal non-tumor surgical specimens from 34 patients diagnosed with gastric adenocarcinoma. Randomly selected samples were obtained from the Iran National Tumor Bank. cDNA synthesis was carried out on total RNA, by using the miRCURY LNA™ Universal RT microRNA PCR Kit. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) assays were performed with specific LNA™ primers and SYBR Green master mix. The human embryonic carcinoma cell line, NTERA2 (NT2) and a human gastric adenocarcinoma cell line, AGS, were used to optimize the PCR reactions. A comparative evaluation of miR-302b expression in tumor and non-tumor gastric samples was performed by either paired t test or Wilcoxon non-parametric test. The ability of miR-302b to discriminate tumor from non-tumor gastric samples was evaluated using the area under the receiver operating characteristic (ROC) curve. According to our data, miR-302b expression (normalized to that of the U6 snRNA housekeeping gene) in the pluripotent cell line NT2 was more than 500 times greater than that of the AGS cell line. The level of expression was even lower in tumor and non-tumor gastric tissue samples. The data further revealed a down-regulation of miR-302b in gastric tumor samples (p=0.001), particularly in high-grade adenocarcinoma (p=0.009). However, ROC analysis data demonstrated a low sensitivity and specificity of miR-302b expression to discriminate between the tumor and non-tumor state of the samples (AUC=0.63). Despite the upregulation of some hESC-specific genes in tumors, our data revealed a down-regulation of miR-302b in high-grade tumors. This data suggested a potential tumor-suppressor role for miR-302b in tumorigenesis of gastric tissue.

Lower pregnancy rates of in vitro matured oocytes compared to those of in vivo stimulated cycles indicate that optimization of in vitro maturation (IVM) remains a challenge. Reduced developmental competence of in vitro matured oocytes shows that current culture systems for oocyte maturation do not adequately support nuclear and/or cytoplasmic maturation. Therefore this study evaluates the effects of different concentrations of saffron (Crocus sativus L.) aqueous extract (SAE), as an antioxidant agent on IVM of immature mouse oocytes. In this experimental study, cumulus-oocyte complexes (COCs) were collected from 6-8 weeks old novel medical research institute (NMRI) female mice ovaries. COCs were cultured in IVM medium supplemented with 0 (control), 5, 10, 20 and 40 μg/ml of SAE in 5% CO2 at 37°C. The rates of maturation, fertilization and development were recorded. ANOVA and Duncan’s protected least significant test, using the SAS program was applied for all statistical analysis. The maturation rate was significantly higher in all groups treated with different concentrations of SAE compared with the control group (p<0.05). However, the lower concentrations of SAE (10 and 5 μg/ml) in maturation medium respectively increased the fertilization rate of oocytes and in vitro developmental competence when compared with the control group (p<0.05). The results of this study indicate that lower concentrations of SAE are more appropriate to be added to maturation medium when compared with other experimental and control groups. Generally, we conclude that addition of appropriate amounts of natural extracts such as SAE to maturation medium improves oocyte maturation and embryo development.

This research study is an attempt to examine whether the administration of ethanol after memory reactivation would modulate subsequent expression of memory in rats. Additionally, we examined whether this administration alters the density of Cornu Ammonis (CA)1 and CA3 pyramidal and dentate gyrus (DG) granule cells. In this experimental study, adult male Wistar rats (200-300 g) were trained in a fear conditioning system using two 1 second, 0.6 mA shocks with an interval of 180 seconds. Twenty four hours later rats were returned to the chamber for 120 seconds. Immediately after reactivation they were injected with ethanol (0.5, 1, 1.5 mg/ kg) or saline. 1, 7 and 14 days after reactivation, rats were returned to the context for 5 minutes. Seconds of freezing (absence of all movement except respiration) were scored. In the second experiment (described in the previous paragraph), after test 1, animals were anesthetized with sodium pentobarbital and perfused transcardially with phosphate buffer (10 minutes) and 4% paraformaldehyde (15 minutes). The brains were postfixed in phosphate-buffered 4% paraformaldehyde (24 hours) and 30% sucrose. 10-μm sections were stained with cresyl violet. Data were analyzed by 1-and 2-way ANOVA for repeated measurements by means of SPSS 16.0. Tukey’s post hoc test was performed to determine the source of detected significant differences. P <0. 05 were considered significant. Data are presented as mean ± SEM. Findings from the first experiment indicated that ethanol at a dose of 1.5 mg/kg significantly impaired recall of memory only in the first test. The density of CA1 and CA3 pyramidal and DG granule cells in the ethanol group was decreased (p< 0.01) compared with control group respectively 43.7%, 35.8%, and 37.8. The data demonstrate that ethanol exposure impairs post retrieval processes. Moreover, ethanol decreases the density of CA1, CA3 and DG cells. Presumably it would be a correlation between our behavioral and histological results.

3,4-methylenedioxymethamphetamine (MDMA) is an illicit, recreational drug that causes cellular death and neurotoxicity. This study evaluates the effects of different doses of MDMA on the expression of apoptosis–related proteins and genes in the hippocampus of adult rats. In this expremental study,a total of 20 male Sprague Dawley rats (200-250 g) were treated with MDMA (0, 5, 10, 20 mg/kg i.p. twice daily) for 7 days. Seven days after the last administration of MDMA, the rats were killed. Bax and Bcl-2 genes in addition to protein expressions were detected by western blot and reverse transcriptionpolymerase chain reaction (RT-PCR).Results were analyzed using one-way ANOVA and p≤0.05 was considered statistically significant. Our results showed that MDMA caused dose dependent up-regulation of Bax and down-regulation of Bcl-2 in the hippocampus. There was a significant alteration in bcl-2 and bax genes density. Changes in apoptosis-related proteins and respective genes relating to Bax and Bcl-2 might be involved in the molecular mechanism of MDMA-induced apoptosis.

Anti-tumor immunity and cytokine profiles have important roles in the development of cancer. Norepinephrine (NE) release due to sympathetic activation leads to a Th2 deviation via the beta-2 adrenergic receptor Beta-2 adrenergic receptor (β-2AR) and could increase cancer progression. This study intends to determine the effects of isoproterenol (ISO; beta-agonist) and propranolol (PRO; beta-antagonist) on the production of IFN-γ, IL-4, and IL-17. Cytokine levels have been examined in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) of patients with colorectal cancer (CRC). The β-2AR expression on lymphocyte subsets was also assessed. In this experimental study, TILs were isolated from fresh CRC tissue and patient PBMCs were obtained just prior to surgery. The cells were cultured in medium for 72 hours. Concomitantly, cells were stimulated with 10 μg/ml phytohemagglutinin (PHA) alone or in the presence of either 1 μmol/L of PRO or 1 μmol/L ISO. The concentration of cytokines in the supernatants was measured by ELISA. Three-color flow cytometry was used to determine the expression of β-2AR on the lymphocyte subsets. Statistical analyses were performed via paired or independent t-test. Levels of IFN-γ, IL-4 and IL-17 were elevated after PHA-stimulation of PBMCs and TILs. However, the elevation of IFN-γ and IL-17 production by TILs in response to PHA was significantly lower than PBMCs. In the presence of ISO, the IFN-γ/IL-4 ratio reduced in all groups, but this reduction was very low in TILs. Interestingly, the effects of PRO on cytokine production were, at least partially, comparable to those of ISO. Depressed levels of β-2AR expression were demonstrated on CD4+IFN-γ+ and CD4+IL-17+ lymphocytes in patients’ PBMCs and TILs. This study has demonstrated the effects of ISO and PRO on cytokine production by TILs and determined β-2AR expression on these cells. ISO failed to induce a shift toward the expected Th2 cytokine profile in CRC patients’ TILs, which might be due to the downregulation of β-2AR expression on TILs. Additionally, in this study, PRO induced a shift to a Th2 profile in PBMCs.