Cell Journal (Yakhteh)
Year:2007 | Volume:9 | Issue:3 (35)|Papers Count:12

The capacity of mesenchymal stem cells (MSCs) to differentiate among skeletal cell lineages and to undergo extensive proliferation in vitro renders them an appropriate source for cell-replacement therapy to heal defects of bone and cartilage tissue.It is argued that in bone and cartilage defects, MSCs would better be transplanted as fully-differentiated cells otherwise they may produce non-specific cells in defect sites. This notion may emphasize the importance of the studies considering in vitro bone and cartilage differentiation of MSCs. Indeed, the capacity of producing osteoblastic and chondrocytic cell lineage is among the earliest differentiation potentials of MSCs, being reported at first isolation of the cells. Recent studies, however indicate that MSCs could differentiate into more cell lineages than expected. The present study provides evidence for, in vitro potential of MSCs to differentiate into bone and cartilage cell lineages. As an introduction to the differentiation, the characteristics of MSCs have been described and MSCs differentiation reviewed. The culture condition for bone and cartilage differentiation, molecular regulation of the differentiation, signaling pathway involved during the differentiation, and the genes up-regulated upon bone and cartilage differentiation have also been described.

Objective: Reporter gene transfer to mammalian cells receives a great deal of attention due to its importance for molecular biology, embryology and developmental biology studies. Among DNA transfer technologies to eukaryotic cells, lipofection is known as the most widely used because of its easy handling procedure, low cell mortality and the natural pathway it undertakes.Materials and Methods: In this study we have examined the transfectability of two cell types: CHO and Vero cells via Lipofection in four different treatments, with combination of exposure duration, 3 and 6 hrs, and different plasmid DNA concentration, 0.5 and 1mgs. A fusion protein expression vector, pUcD2. PTS2-EGFP was used to direct the EGFP protein to peroxisomes after expression of related cDNA. An SPSS analysis was preformed after counting the positive cells.Results: optimum gene expression was found when using 1mg DNA treated for three hrs for CHO cells, and 1mg DNA treated for six hrs for Vero cells.Conclusion: The result suggests that CHO lipofection efficiency is significantly increased by both the DNA concentration and exposure time increment; however, an increase in exposure time has less significant effect on low DNA concentration conditions. The same results have been observed for Vero cells. Optimum expression was obtained with highest DNA concentration.

Objective: Embryonic stem cells (ESCs) are pluripotent cells capable of extensive proliferation while maintaining their potential to differentiate into any cell type. The therapeutic potential of these cells are promising, but in many cases limited by our inability to promote their differentiation. In this study we examined the effect of inducer factors in direct differentiation of mouse ESCs into a neural fate in suspension culture systems.Materials and Methods: Mouse ESCs (Royan B1) were cultivated in suspension to form embryoid bodies (EBs) within 2 days (2d). They were induced using astrocyte-conditioned medium (ACM), retinoic acid (RA, 1mM) and basic fibroblast growth factor (bFGF, 20 ng/ml) for 4 days (2+4d) and were cultured on poly-L-lysine coated dishes up to 5 days (2+4+5d) to differentiate. The expressions of neural specific genes were analyzed by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Differences of means for percentage significance of differentiated EBs were tested by the Mann-Whitney Test and for length and thickness of neural processes by t-test.Results: Retinoic acid increased the percentage of EBs outgrowth with neural morphology and bFGF had synergistic effect (at least p<0.05), while ACM did not influence neural differentiation at all. Immunofluorescence analysis revealed the expression of b-tubulin III in neural cells and that the differentiated neural cells in RA+bFGF group were longer and thicker processes (at least p<0.05). The cells expressed Nestin, Pax6, NF-M, Islet-1, Lim1, and HB9.Conclusion: The results showed that ACM had no effect on neural differentiation from ESCs, however, RA was in favor of this and that bFGF increased neural differentiation synergistically by mechanisms that remain to be defined.

Objective: The purpose of the present study was to verify the ultrastructural characteristics of cultured mouse preantral follicles from fresh and vitrified –warmed ovarian tissues.Materials and Methods: The ovaries of 14-day-old mice (n=5) were vitrified using a mixture of ethylene glycol, ficoll 70 and sucrose. The preantral follicles were isolated and cultured in a-minimum essential medium supplemented with 5% fetal bovine serum, 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin and selenium, 20ng/ml murine recombinant epidermal growth factor for four days. The follicles were fixed in 2.5% glutaraldehyde and post fixed with 1% osmium tetroxide and processed for transmission electron microscopic studies.Results: Cultured preantral follicles of the control group on day 4 consisted of an oocyte surrounded by several layers of polyhedral granulosa and flattened theca cells. The granulosa cells and oocytes were in close connection. Several granulosa cell projections could be seen in the outer layer of zona pellucida. Polyribosomes and rough endoplasmic reticulum cisternae were observed in the most samples in association with mitochondria. The ultrastructure of vitrified follicles was similar to the control. The homogenous zona pellucida surrounded the oocytes but the granulosa cell projections did not enter it. The subzonal space was wider than that of the control group. Conclusion: Ovarian vitrification and subsequent in vitro growth, in vitro maturation and in vitro fertilization with minimal ultrastructural changes in oocyte could be an alternative to preserve fertility of infertile women.

Objective: This study was performed to determine the immunophenotype of Megakaryocyte progenitor cells differentiated from UCB CD133+ and CD133− cells under the effects of interleukin–3 (IL–3), interleukin–6 (IL–6), stem cell factor (SCF) and thrombopoietin (TPO) in vitro.Materials and Methods: CD133+and CD133− cells were isolated by using CD133 isolation kit following the manufacturer’s instructions. Then, they were seeded in liquid serum free expansion mediums supplemented with the cytokine cocktail including IL–3, IL–6, SCF and TPO. The expression rate of CD34, CD41, CD61 and CD42b were measured on the days 0 and 7 of culture using flow cytometry. Student’s t–test was used for the comparisons and a p value less than 0.05 was considered to be significant.Results: Expressions of megakaryocytic markers on CD133+ cells were always higher than CD133− cells. CD133+ cells have higher potential of generating Mk colonies in vitro.Conclusion: CD133+ subset may be used as an alternative source for Mk progenitor cells production and these cells may improve platelet recovery after UCB transplantation.

Objective: Although Iron is a crucial element for many metabolic pathways of the body, the excess iron may induce apoptosis in some cell types such as macrophages. In the present investigation, the role of iron overload in inducing apoptosis of Balb/c mice macrophages infected with L. major in vitro, as a selective model, was studied.Materials and Methods: The peritoneal macrophages were harvested and cultured with different concentrations of iron to refuse its cytotoxic effect. Then, the macrophages were harvested and cultured with or without Leishmania in the presence of iron or donated reagent [S-Nitro-N-Acetylpenicillamine (SNAP)] or an inhibitor of NO, synthase [NG-Methyl-L-Arginine (NMMA)]. The concentration of NO as an immunological mediator in culture supernatants was measured after 18 hour incubation. Simultaneously, macrophages undergoing apoptosis were identified by fluorescence and electron microscopy. Results: The findings showed that there is a statistically significant relationship between iron overload and apoptosis (p<0.05). Apoptosis rate had also increased in the macrophages cultured with iron, SNAP, NMMA, as compared with control group.Conclusion: Iron influences the apoptosis rate and NO production in the macrophages infected with L. major.

Objective: Polymorphism of the size of heterochromatin region of chromosomes has been well documented in human genome and it consists of DNA sequences that are not transcribed. The prime aim of the present study was to evaluate the heterochromatin polymorphism associated with chromosomes in leukemic patients.Materials and Methods: The study was conducted on 35 consecutive leukemic patients and 34 healthy individuals in Modaress and Taleghani hospitals, Tehran, Iran between 2004-2006. By applying Barium Hydroxide saline Giemsa (BSC) method with certain alterations, the variant heterochromatin polymorphism of chromosomes 1, 9 and 16 on bone marrow and peripheral blood lymphocyte cultures were evaluated. Chi-square and Fisher’s exact tests were used for statistical analysis with SPSS software.Results: Constitutive heterochromatin polymorphism of chromosomes 1 and 9 in leukemic patients revealed statistical significant differences when compared with chromosomes of healthy controls (p=0.0005) and (p=0.006) respectively. The differences were not significant for chromosome 16, it was 11.4% in leukemic patients and 0% in the control group (p=0.05). The frequency of partial and complete inversions did not show any significant differences between the leukemic patients and the control group. Conclusion: The constitutive heterochromatin polymorphism blocks may provide an opportunity to serve as a marker for the detection and characterization of the chromosomes in leukemic patients.

The dorsal skin of the Iranian frog, Rana ridibanda, is associated with numerous prominent granular glands which extract their secretions in the response to stressor or invading pathogens .The secretions have broad spectrum antimicrobial effects. In this research the effect of antimicrobial skin secretions from Iranian frog (Rana ridibanda) has been examined against gram positive Methycillin Resistant Staph Aureus (MRSA), under sterile conditions. To show this effect, 1cm2 of frog skin was cultured in complete tissue culture medium containing RPMI, FBS and FUNGISON for the period of 10 days. Immediately after, MRSA was exposed to frog skin secretion (medium culture) and Vancomycin. The results showed that the frog skin secretions has significant antimicrobial effect against MRSA. The range of inhibition zone for MRSA was the same as (20mm) Vancomycin in DISK method. In Minimum Inhibitory Concentration method, for MRSA, the tube 1/8 was positive.