Cell Journal (Yakhteh)
Volume:12 Issue: 2, 2010

Spermatogonial stem cells (SSCs) are in the beginning of a complex process in which they transmit genetic information from generation to generation. Any failure in this process can result in infertility. It has been suggested that transplantation of spermatogonial stem cells, following their maintenance and culturing, may restore fertility in some infertile patients. Because fertility restoration through SSCs transplantation has been successfully achieved in animal experiments, we hope human studies can follow in the near future. The isolation and cultivation of SSCs help us study their biological characteristics and their application in therapeutic approaches. In this review, we studied spermatogenesis in rodents and humans. We also compared markers and different SSC culture systems in both.

The aim of this study was to investigate the preventive effect of the polyamine component N, N, N´, N´-tetrakis(2-aminoethyl)2,2-dimethylpropane-1,3-diamine (tdmtn) on apoptotic motor neurons in adult mouse spinal cord slices. Thoracic region of adult mouse spinal cords was sliced by a tissue chopper into 400 μm slices and cultured in medium in the presence or absence of tdmtn for 6 hours. Morphological a features of apoptosis were evaluated using fluorescent staining with propidium iodide and Hoechst 33342. The appearance of nucleosomal DNA fragmentation was studied using agarose gel electrophoresis. Our results were analyzed using the one-way ANOVA and Tukey’s tests. After 6 hours in culture, motor neurons displayed morphological sings of apoptosis including cell shrinkage, as well as nuclear and chromatin condensation. DNA extracted from slices cultured for 24 hours revealed nucleosomal DNA fragmentation on agarose gel electrophoresis. Tdmtn reduced the occurrence of apoptosis in motor neurons and significantly (p<0.01) increased the viability of these neurons after 6 hours of culturing. Tdmtn, as a polyamine component, could probably prevent the occurrence of motor neuron apoptosis through calcium chelation.

Artificial stimulation of mouse oocyte, in the absence of sperm contribution, can induce its parthenogenic activation of oocyte. Ultrasound is one of the newest methods for artificial activation of mammal oocytes, and its successful utilization in pig oocyte activation has been recently reported. Our objective was to assess the effect of ultrasound on mouse oocyte activation. Our groups included1 control group, 3 experimental groups consisting of 1, 2 and 3 repetitions of ultrasound exposure, and 3 sham groups handled similar to experimental groups but ultrasound system was off during treatments.In experimental groups, adult female NMRI mice at the interval between pregnant mare serum gonadotropin (PMSG) and human corionic gonadotropin (hCG) injections, were exposed to continuous ultrasound with 3.28 MHz frequency and peak intensity (Ipk) = 355 mW/cm2.Sixteen hours after injection of hCG, the mice were euthanized and their oocytes were collected; thereafter, parthenogenic oocytes were counted. Data analysis using the ANOVA test shows a significant increase in the number of parthenogenic oocytes in mice with 3 overall exposures to ovarian ultrasound (p<0.05). A significant decrease in the number of metaphase II (MII) oocytes numbers was also seen in mice treated with ultrasound (p<0.05). Ultrasound is thought to induce pores generation in oocyte membranes and provides an easier inward transport of Ca++ into oocytes. This phenomenon can induce meiosis resumption in immature oocytes. With increased exposure repetitions from 1 to 3 times and greater Ca++ arrival, oocytes can be parthenogenetically activated.

The aim of this study is to create an ex vivo model to examine the expression of two heat shock protein 70 (HSP70) family members, heat shock protein 72 (HSP72) and heat shock constitute protein 70 (HSC70), at the mRNA and protein levels in differentiating corneal cells from air exposed limbal stem cells. Limbal biopsies were cultured as explants on a cellular amniotic membrane for 14 days. The cells were then exposed to air for 16 extra additional days. The proposed expression of limbal stem cell markers (p63, ABCG2), corneal markers (K3/12, connexin 43), as well as HSP72 and HSC70 were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) at the mRNA level, and by immunocytochemistry and flowcytometry at the protein level both pre and post air exposure. Fresh limbal and corneal tissues were used as control group. Air exposure decreased expression of p63 and increased expression of K3/K12 indicating an increase in the number of corneal cells. Our data showed that HSP72 and HSC70 were expressed at the mRNA level before and after air exposure while their expression significantly increased post air exposure at the protein level. We assume HSC70 expression may be related to early and terminal stages of differentiation in cultured limbal stem cells. In addition, limbal stem cells were protected during normal development against oxidative stress thru increased HSP72 expression. These findings may have broader implications in development of therapeutic strategies for treating wound healing disorders by induction of HSPs.

Considering that cannabinoids protect neurons against neurodegeneration, in this study, the neuroprotective effect of WIN55,212-2 in paraoxon induced neurotoxicity in PC12 cells and the role of the N-methyl-D-aspartate (NMDA) receptor were evaluated. In this study PC12 cells were maintained in Dulbecco's modified eagle’s medium (DMEM+F12) culture medium supplemented with 10% fetal bovine serum. The cells were treated with paraoxon (200 μM) in the presence or absence of WIN55,212-2 (0.1 μM), NMDA receptor agonist NMDA (100 μM), cannabinoid receptor antagonist AM251 and NMDA receptor antagonist MK801 (1 μM) at 15 minutes intervals. After 48 hours of exposure, cellular viability and protein expression of the CB1 receptor were evaluated in PC12 cells. Following the exposure of PC12 cells to paraoxon (200 µM), a reduction in cell survival and protein level of the CB1 receptor was observed (p<0.01). Treatment of the cells with WIN55,212-2 (0.1 µM) and NMDA (100 µM) prior to paraoxon exposure significantly elevated cell survival and protein level of the CB1 receptor (p<0.01). Also, AM251 (1μM) did not inhibit the cell survival and protein level of the CB1 receptor increase induced by WIN55,212-2 (p<0.001). However, MK801 (1 μM) did inhibit cell survival and protein expression of the CB1 receptor increase induced by NMDA (p<0.001). The results indicate that WIN55,212-2 and NMDA protect PC12 cells against paraoxon induced toxicity. In addition, the neuroprotective effect of WIN55,212-2 and NMDA was cannabinoid receptor-independent and NMDA receptor dependent, respectively.

The aim of this study was optimization of the PolyFect gene delivery method of pcDNA3.1 expression vector transfected with the mouse pdx-1 gene in three different kinds of mesenchymal stem cells and Hepa cells as well as comparison of transfection efficiency leading to expression of the mentioned gene in the cell types used. Rat bone marrow-derived mesenchymal stem cells, C57 mouse bone marrow-derived mesenchymal stem cells, human synovium derived mesenchymal stem cells and Hepa cells were used in this study. After culturing of the mentioned cells, mouse pdx-1 gene were transfected into them using the Qiagen PolyFect kit. 72 hours later, the cells were treated with anti-mouse Pdx-1 antibody and immunocytochemically analyzed using a fluorescent inverted microscope. Transfection conditions were optimized in each of these cells by changing different lipofection parameters such as DNA concentration, PolyFect reagent concentration and cell density. The results demonstrated that for transfection of these cells, the best concentrations of DNA and PolyFect reagent are 400 ng/µL and 6000 ng/µL respectively. For maximum transfection efficiency, the best cell density in 12-well plates was 105 cells in Hepa cells, 1.3×105 cells in rat bone marrow-derived mesenchymal stem cells, 1.5×105 cells in human synovium-derived mesenchymal stem cells and 105 cells in C57 mouse bone marrow-derived mesenchymal stem cells. Under the mentioned optimized conditions, the maximum efficiency of transfection was determined to be 50% for Hepa cells, 40% for rat bone marrow-derived mesenchymal stem cells, 21% for human synovium-derived mesenchymal stem cells and 10% for C57 mouse bone marrow-derived mesenchymal stem cells. These findings implicate that the most important factor extremely influencing transfection efficiency in mesenchymal stem cells is the cell derivation origin. Results of this study can be used in basic and clinical studies dealing with gene therapy in mesenchymal stem cells.

Replacment of CD133+ cell’s β globin gene by using a gene targeting construct containing the β globin gene and essential elements for homologous recombination. pFBGGT was amplified, then digested using the NheI and XhoI restriction enzymes, and finally, a 13.3 kb band (naked DNA) was extracted from the agarose gel. Biological activity of positive and negative selection markers were checked by transfection of COS-7 cells with linear plasmid. Hematopoietic stem cells (HSCs) were separated and transfected with linear plasmids using lipofection followed by positive and negative selection. Polymerase chain reaction (PCR) were done on DNA from the selected cells and the products were sequenced. The results of biological activity assays showed that selection markers were active. PCRs for hygromycin, neomycin and joining segments were positive but PCRs for TK1 and TK2 genes were negative. Sequencing PCR product joining segment confirmed the formation of homologous recombination. In this novel strategy gene replacement was achieved and biological activities of its components were observed.

The aim of this study was to produce a stable CHO cell line expressing tenecteplase. In the first step، the tenecteplase coding sequence was cloned in a pDB2 vector containing attB recognition sites for the phage φC31 integrase. Then، using lipofection، the CHO cells were co-transfected with constructed recombinant plasmid encoding tenecteplase and attB recognition sites and the integrase coding sequence containing pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistance gene (neo)، stable cells were then selected using G418 as an antibiotic. Stable transformed cells were assessed using genomic PCR and RT-PCR. Finally، the functionality of tenecteplase was evaluated on the cell culture media. our results indicated that tenecteplase coding sequence was inserted into the CHO cell genome and was successfully expressed. Moreover، tenecteplase activity assessment indicated the presence of our functional tenecteplase in the cell culture medium. Considering the data obtained from this study، φC31 integrase can be used for the production of a stable cell line and it be used to introduce ectopic genes into mammalian cells.

Dendritic cells (DCs), as the managers of the immune response, have a crucial role in forming the direction and nature of the immune response. Some compounds such as 1,25-dihydroxycholecalciferol affect the function of DCs and can be used to shift the immune functions toward favorite directions. The aim of this study was to investigate the in vivo effects of 1, 25- dihydroxycholecalciferol on DCs surface markers, their potential to induce specific T-cell responses and the cytokines profile. 1, 25-dihidroxycholecolciferol was regularly injected intraperitoneal into C57BL/6 mice. DCs were separated from the spleens of calciferol treated and non-treated mice using magnetic beads. The expression of DCs surface markers was investigated by flow cytometric analysis. The separated cells were pulsed by myelin oligodendrocyte glycoprotein (MOG) and injected subcutaneously into front footpads of syngeneic mice. After five days, the lymphocytes from regional lymph nodes were separated and used for the lymphocyte transformation test (LTT) and determination of the interferon gamma/interleukin 4 (IFNγ/IL-4) ratio by ELISA technique. Statistical analysis of the obtained results showed reduced expression ofmaturation markers and co-stimulatory molecules by cholecalciferol treated DCs. Thespecific T-cell stimulation potential of treated DCs as well as the induced IFNγ/IL-4 ratiowas also down-regulated compared to non-treated cells (p value<0.05). It seems that 1,25-dihydroxycholecolciferol can regulate the DCs function and maturation state in vivo. The T-cell stimulation rate and Th1/Th2 cytokines ratio also changes following interaction with cholecalciferol treated DCs.

Evaluating the expression of Oct-4، NANOG، Sox2 and Nucleostemin in colon cancer cell lines (Caco-2 and HT-29). Caco-2 and HT-29 human colon cancer cell lines were cultured in Dulbecco’s modified eagles medium (DMEM) and Roswell Park Memorial Institute medium (RPMI) respectively، containing 10% fetal bovin serum (FBS) with 1% peniciline and streptomycinen in 37°، 5% CO2 incubator. Total RNA was isolated using the ISOGEN method. RNA integrity was checked with the use of agarose gel electrophoresis and spectrophotometry. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to examin the samples. The expression of Oct-4 and Nucleostemin at the protein level wasfurther determined using immunocytochemistry. RT-PCR analysis of Caco2 and HT-29 colon cancer cell lines showed expression of Oct-4، NANOG، Sox2 and Nucleostemin genes. Also immunocytochemical analysis confirmed the cytoplasmic and nuclear expression of the Oct-4 protein and Nucleostemin proteins. Collectively، our data confirmed the expression of Oct-4، NANOG، Sox2 and Nucleostemin in colon cancer cells and suggested that their expression can be used as potential tumor markers in diagnosis and /or prognosis of colon tumors. These results confirm the potential value of the cancer stem-cell theory in cancer therapy.

The aim of this study was to compare the colony formation of spermatogonial stem cells (SSCs) on sertoli and STO (Mouse embryonic fibroblast cell line) feeder cell layers during a two-week period. Initially, sertoli cells and SSCs were isolated from adult mouse testes using a two-step enzymatic digestion and lectin immobilization. Characteristics of the isolated cells were immunocytochemically confirmed by examining for the presence of Oct-4, CDH1, promyelocytic leukaemia zinc finger factor (PLZF), SSC C-kit, and the distribution of Sertoli cell vimentin. SSCs were then cultured above the Sertoli, STO and the control (without co-culture) separately for two weeks. In all three groups, the number and diameter of colonies were evaluated using an invert microscope on the 3rd, 7th, 10th and 14th day. β1 and α6-integrin m-RNA expressions were assessed using a reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. Furthermore, Oct-4 m RNA expression was assessed using real time PCR. Statistical analysis was performed using ANOVA; and the paired two-sample t test and Tukey’s test were used as post-hoc tests for the data analysis of the three sertoli, STO and control cocultures. At the four specified time points, our results showed significant differences (p<0.05) in colony numbers and diameters among the sertoli, STO and control groups. The number and diameter of colonies increased more rapidly in the sertoli coculture than in the other two Our results at all four time points also showed significant differences (p<0.05) in the mean colony numbers and diameters between the three groups, with the Sertoli coculture having the highest mean values for colony numbers and diameters. The RT-PCR results, after two-weeks of culturing, showed that β1-integrin was expressed in all three groups cocultures, but α6-integrin was not expressed. Additionally, based on real time PCR results, the three genes (β1-integrin, α6-integrin, Oct-4) mentioned were also expressed in all three co cultures groups. Based on the optimal effects of sertoli feeder cells on spermatogonial stem cells in a co culture system, as also confirmed by several other studies, their use is suggested to achieve better colonization of SSCs.

Chronic myeloid leukemia (CML) develops when a hematopoietic stem cell acquires the BCR/ABL fusion gene. This causes these transformed hematopoietic cells to have a greater than normal proliferation rate. Scientists attempt to improve the CML treatment process by silencing the BCR/ABL oncogene. In this work, we used morpholino antisense oligos to silence the BCR/ABL oncogene. In this study, the K562 was used as a BCR/ABL fusion-gene positive cell line and the Jurkat cell line as a control. We explored the inhibiting capacity of morpholino antisense oligos in the the expression of the BCR/ABL oncogene and studied their p210 BCR/ABL suppression, inhibition of cell proliferation and stimulation of apoptosis in the K562 cells after 24 and 48 hours. Endo-Porter was used for delivery ofmorpholino antisense oligos into cell cytosols. Meanwhile, flow cytometric analysis wasperformed in order to determine the appropriate concentration of morpholino antisenseoligos. Prolonged exposure of the K562 cell line to the morpholino antisense oligos targeted against the BCR-ABL gene showed proliferation inhibition as its main feature. After western blotting, we found that complete silencing of BCR/ABL was achieved, but flow cytometric analysis showed no broad apoptosis. The results indicate that the Morpholino antisense oligo is able to inhibit p210 BCR/ABL; however, it cannot induce broad apoptosis due to co-silencing of BCR.

Rapid diagnosis of Trisomy 21 Syndrome (Down Syndrome) patients using Real-Time quantitative Polymerase Chain Reaction (Real-Time qPCR) in order to establish a novel method for prenatal diagnosis in the future. A total of 5 ml of peripheral blood was obtained from each patient and normal controls (NR). Then, genomic DNA from lymphocytes was extracted using the salting out procedure. Gene dosage levels of DSCAM and (PMP22, DSCAM) in Down Syndrome and NR were analyzed using real-time quantitative PCR. The DSCAM/PMP22 ratio was calculated according to the 2-ΔΔCt formula for all samples. Real-time PCR showed a DSCAM/PMP22 ratio of 1.48 ± 0.18 and 1.01 ± 0.10 (p<0.001) in Down Syndrome and normal samples, respectively, demonstrating three copies of the target (DSCAM) gene in Trisomy 21 Syndrome. DSCAM/PMP22 ratio is increased significantly in Down Syndrome patients than NR (1.5 times). Therefore, the real-time quantitative PCR technique can be used as a sensitive, accurate and reliable technique for rapid and prenatal diagnosis of Trisomy 21 Syndrome.

The purpose of this study was to evaluate the quantitative expressions of BAG1, BAX and BCL-2 in human embryos with different fragmentation grades as derived from assisted reproduction technology (ART). Fragmented and normal human 8-cell embryos were scored according to the degree of fragmentation with an inverted microscope and divided into four grades (grade І: no or minimal fragmentation (<5%), grade ІІ: embryos with <25% fragmentation, grade ІІІ: embryos with >25% fragmentation and grade ІV: apoptotic induced embryos with actinomycin D). In this study, TUNEL labeling was initially used to detect apoptosis, and then revers transcription polymerase chain reaction (RT-PCR) and quantitative PCR were used to define the quantitative expressions of experimental genes in human embryos with different fragmentation grades. The results of TUNEL labeling showed that embryos with higher fragmentation had a high number of apoptotic bodies. The results of RT-PCR and q-PCR analyses showed a significantly decreased amount of BAGI transcript expression from group I to group IV. The highest expression of BAX gene was observed in group II, however, the transcript of BCL-2 gene was not observed in any of the experimental groups. The effect of actinomycin D on transcript expression amounts of experimental genes in apoptotic induced embryos (group IV) compared to control embryos (group I) showed a significant decrease. mRNA expression of BAG1 gene can be used as a good marker to detect apoptosis in human embryos. However, the transcript of BCL-2 gene does not play a role in the detection of apoptosis in human embryos at the 8-cell stage.

It has been shown that lithium chloride (LiCl), an effective drug for the treatment of bipolar disorder, has side effects on the female reproductive system. In this study, cellular and histological effects of lithium chloride on the development of ovarian follicles in immature female rats were investigated. To induce ovarian follicular development, twenty-three day old immature female rats were injected with 10 IU of pregnant mare serum gonadotropin (PMSG), followed by four doses of LiCl (250 mg/kg/dose) each injected every 12 hours, starting from the time of the PMSG injection. The rat ovaries were removed 48 hours after the PMSG injection and prepared for histological, immunohistochemical, and DNA laddering studies. Control immature female rats received only PMSG, while sham treated rats received PMSG and physiological serum (lithium vehicle). Our results showed that in the ovaries of LiCl-treated rats there were neither large antral follicles (800-1000 µm) nor fewer medium sized follicles (400-800 µm) but a increased number of atretic follicles compared to those in the control rats. The induction of atresia in the ovaries of LiCl-treated rats was further confirmed by the presence of DNA fragmentation. Looking at the cellular levels, lithium extremely significant (p<0.0001) increased the number of TUNEL-positive cells in the granulosa layer of the antral follicles. Taken together, our results suggest that lithium may decrease folliculogenesis by inducing apoptosis in the antral follicles.

To study the impact of ultraweb nano-fibrillar substrates on in vitro differentiation of mouse bone marrow-derived mesenchymal stem cells into hepatocyte-like cells.

For a duration of up to 36 days, mouse bone marrow mesenchymal stem cells (MSCs) were cultured on an artificial basement membrane containing ultraweb nanofiber (nano+) and plates (nano-) coated with Matrigel and used growth and differentiating factors for hepatogenic differentiation. Then, to study the impact of nanofibers on hepatocyte differentiation, mRNA expressions for liver-specific genes (CK7, AFP, FOXa2, ALB, CYP1a1, HNF4a) were analyzed using real-time PCR, and ultrastructures of differentiated cells were evaluated using scanning and transmission electron microscope.

Both experimental groups expressed mRNA of liver-specific genes in a time dependent manner. However, mRNA expression trends of liver-specific genes, especially those of FOXa2 and HNF4a, were better in the nano+ culture as compared to the nano- culture (p<0.05). Furthermore, the ultrastructures of differentiated cells in the 36 day-old nano+ culture were more mature as compared to those of the nano- group.

The results suggest that topographical properties of the extracellular matrices produced by ultraweb nanofibers are effective for the in vitro reproduction of more differentiated MSC-derived hepatocyte-like cells.

In recent years, studies have focused on the telomerase for cancer treatment by repressing telomerase in cancerous cells or prevent cell aging by activating it in the aged cells. Thus, in these studies natural and synthetic agents have been used to repress or activate telomerase. In this research, we investigated the effects of human growth hormone (hGH) on aging via evaluation of telomerase activity. Primary human foreskin fibroblast cells were isolated, cultured and treated with different concentrations of hGH. BrdU and MTT cell proliferation assays and cells number counting. Cell aging was assayed by the senescence sensitive galactosidase staining method. Telomerase activity was measured with a telomerase PCR ELISA kit.Data were analyzed with SPSS software (one-way ANOVA and univariate ANOVA). Our results indicated that cells treated with a lower concentration (0.1, 1 ng/ml) of hGH had more green color cells (aged cells). Furthermore, cell proliferation increased with increasing hGH concentrations (10 to 100 ng/ml) which was significant in comparison with untreated control cells. TRAP assay results indicated that telomerase activity increased with increasing hGH concentration, but there was no significant difference. Additionally, more rapid cell growth and telomerase activity was noted in the absence of H2O2 when compared with the presence of H2O2, which was significantly different. Although increasing cell proliferation along with increasing hGH concentration was confirmed by all cell proliferation assays, only the cell counting test was statistically significant. Thus, it is inconclusive that hGH (up to 100 ng/ml) has an anti-aging effect. Also, because there was no significant difference in the telomerase activity results (in spite of increasing progress along with increasing hGH concentration) we can not certainly conclude that hGH (up to 100 ng/ml) impacts telomerase activity.