Cell Journal (Yakhteh)
Year:2008 | Volume:10 | Issue:1 (37)|Papers Count:9

The success rate of several advanced basic and clinical techniques in the field of mammalian biotechnology, including cloning, pre-implantation genetic diagnosis, and assisted reproductive techniques (ART) depends mainly on the success rate of pregnancy following in vitro fertilization-embryo transfer (IVF-ET). The techniques used in ART have advanced considerably since the first in vitro fertilization birth in 1978. However, despite these advances, pregnancy rates are still relatively low and have not increased significantly in the last decade. Based on the facts that embryo implantation is considered as the last barrier in ART and that inadequate endometrial receptivity is responsible for approximately two-thirds of implantation failures, intensive research work has been performed to understand the physiology, regulation, and the clinical assessments of the endometrial receptivity to improve the success rate of IVF-ET. This and the ongoing reviews tend to cover the different aspects of the endometrial receptivity mainly in human model. The present part of this series primarily concerns with biochemical and molecular events in the endometrium coordinated within its receptivity period termed as the window of implantation. Successive sections will deal with its ultrastructural changes, biomarkers, clinical assessments and regulators of endometrium within the window of implantation.

Objective: This study was performed to determine whether melatonin at physiological concentrations (0.01-10nM) could affect the proliferation and osteogenic differentiation of Rat ADSCs in vitro.Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced on a monolayer culture with osteogenic medium with or without melatonin at physiological concentrations (0.01-10nM). After 4 weeks cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity by ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium assay and flowcytometry, respectively. All assays were performed in triplicate.Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to the osteogenic medium alone. Similarly, the mineral deposition (calcium level) also decreased. The flow cytometry proved that the cell growth decreased and the apoptotic cells increased.Conclusion: These results suggest that physiological concentration of melatonin has a negative effect on ADSCs osteogenesis.

Objective: CD133+ umbilical cord blood cells were identified as a hematopoietic stem cell which has the capacity for extensive self-renewal and differentiation. The aim of this study was to identify the effect of staurosporine (STS), a well-known protein kinase inhibitor on differentiation of CD133+ cells into neural cells.Materials and Methods: CD133+ cells were enriched by immunomagnetic beads from human mononuclear cells of umbilical cord blood and the purity of higher than 94% was achieved by flowcytometry. Induction of differentiation was performed by addition of STS (12.5, 25, and 50nM). The differentiated cells were evaluated by immunofluorescence and RT-PCR for neuron-specific proteins and transcripts.Results: STS-treated CD133+ cells expressed mRNA transcripts for neuron-specific neurofilament protein (NFM), and several basic helix-loop-helix (bHLH) transcription factors important for early neurogenesis, including Otx2, Wnt1, and Hash1. The structural proteins characteristics of neurons including b-tubulinlll and Microtubule-Associated Protein-2 (MAP-2), were shown by immunocytochemistry. STS-treated CD133+ cells also expressed the astrocyte-specific marker, glial fibrillary acidic protein (GFAP) by immunofluorescence.Conclusion: The human cord blood-derived CD133+ hematopoietic stem cells could differentiate into neural cell types of neuron-like cells and astrocytes by STS treatment.

Objective: The objective of this research was to determine the prevalence of genital C. trachomatis infection in asymptomatic women by using highly sensitive nested-polymerase chain reaction (PCR) in urine sample.Materials and Methods: One hundred-forty asymptomatic women were randomly selected from those who attended gynecology out patient department of Hazraat e Rasool Hospital in Tehran. First catch urine specimen were collected from all the participants. DNA extraction was performed by means of High Pure PCR Template Preparation Kit (HPPTP) according to the manufacture’s instructions. Extracted DNA was tested by omp1 gene based nested-PCR, using sets of primers to amplify C. trachomatis omp1 gene. Visualization of a 1027 bp fragment from omp1 gene in agarose gel electrophoresis was considered as a positive result.Results: In total, 140 urines were tested for determination of C. trachomatis infection. C. trachomatis omp-1 was detected in 22.1% of cases (31/140). The overall prevalence rates of C. trachomatis in the urine sample as determined by omp1 based nested-PCR were 4.3% in group I (age, <25 years), 12.1% in group II (age, 25-34 years), 5.0% in group III (age, 35-44 years) and 0.7% in group IV (>44 years). The highest prevalence of C. trachomatis infection (12.1%) was seen in women aged 25-34 years. This finding was not statistically significant (p=0.710). Also, there was not relation between C. trachomatis infection and some probable risk factors such as young age (<25 years), STD history and missing use of barrier contraceptive in this study.Conclusion: The prevalence of C.trachomatis infection in the women not seeking health care warrants more comprehensive study using high sensitive omp1 based nested- PCR to identify and treat a large number of infected women in Iran.

Objective: To study the effects of different matrices and co-culture systems on cultured limbal stem cells (LSCs).Materials and Methods: Limbal explants were co-cultured with limbal fibroblasts (LF) and/or mouse embryonic fibroblasts (MEF) on filter inserts coated with amniotic membrane (AM), matrigel (MAT) and collagen type I (COL).Results: This study revealed that AM facilitated the cell migration and expansion significantly in comparison with other matrices. However, the gene expression profile of stemness markers of LSCs showed no significant differences among the experimental groups. The data indicated that at least in two-dimensional culture systems, the mentioned matrices have no significant effect on switching the expression of genes involved in differentiation process. In addition, the results of the two co-culture systems in case of different feeders, including MEF and LF were similar in growth rate and also preserving stemness quality of cultured limbal cells.Conclusion: To exclude the pollution of transplantable cultivated cells with probable mouse viruses, LF with human origin is recommended as feeder. Hence, limbal explants grown on AM in co-culture with LF will promise a quick and safe model for preparing undifferentiated epithelial sheets suitable for transplantation.

Objective: Iododeoxyuridine-induced Radiosensitivityi (IUdR) is a halogenated thymidine analogue recognized to be effective in vitro and in vivo radiosensitizer in human cancers. It is reported that Methoxyamine (MX) potentiates DNA damages in cancer cells with blocking the repair pathway of IUdR damages. But studies, entirely, are restricted on monolayer culture cells from human colon cancer cells. Spheroids are 3D form of cells that aggregate and grow together which resemble in vivo tumor models in several aspects and the results of such studies can be extended to tumor in vivo. The aim of the current study was to evaluate DNA damages from IUdR and gamma rays with and without Methoxyamine in human Glioblastoma spheroids.Materials and Methods: The DNA induced damages in U87MG cell line were compared using alkaline comet assay method. Experiments were performed with two different sizes of spheroids (100mm and 300mm).Results: Evaluation of the effects of IUdR with and without MX pretreatment on spheroids following ionizing radiation showed that MX increased the cell damages of IUdR with and without irradiation in both diameters spheroids. The damages were further increased in 100mm compared with 300mm diameter.Conclusion: Comparisons of tail moments in spheroids with 100 and 300mm diameter showed that cell damages in larger spheroids, 300mm, are lesser than smaller one, 100mm. This could be due to existence of G0 cells and cells with longer cycle which IUdR was less incorporated into them. Thus, decrease in IUdR radiosensitization and base wxcision repair (BER), results in reduction of MX activities. Using agents for inhibiting the activities of proteins which are responsible for carrying the cells to G0 may be beneficial in solving such problems.

Objective: In all protocols for isolation of mesenchymal stem cells (MSCs), a few days after culture initiation, the medium were discarded along with its contents of non-adherent cells and the adherent cell population kept and expanded as MSCs population. In the present study, attempt was made to expand the cells suspended in removed medium of primary culture and compare them with the adherent cell population.Materials and Methods: Four days after rat’s bone marrow culture initiation, medium of the culture was collected and its suspended cells were culture-expanded in parallel with adherent cells till passage 3. During the culture period, the cells from either group were statistically compared with respect of the time required for cell confluency (the stage in which cells cover the entire surfaces) as an index of growth rate. At the end, the cells from both cultures were evaluated in terms of their differentiation potential.Results: The primary culture of the cells from removed medium contained large colonies of spindle-shaped cells that reached into confluency after 5.36±0.5 days, while those from the adherent population possessed small colonies reaching into confluency in 8.09±0.70 days. According to the results, at all studied passages, the cells of removed medium were significantly (p<0.05) achieved confluency in shorter time than the adherent population. Moreover, the cells from either culture could easily differentiate into bone, cartilage and adipose cells.Conclusions: It seems that some cells from removed medium, usually discarded in medium substitution, are MSCs possessing more growth rate than the primarily adherent cell population.

In this study, chondrocyte culture was established for the first time in Iran, and calcium alginate was used for longer culture of chondrocyte in vitro. The study was programmed in order to be used for future human chondrocyte transplantation. The cartilage specimen obtained from 50 patients who underwent total knee and hip operations in Isfahan University of Medical Sciences. Cartilage specimens were used for monolayer as well as suspension culture in alginate beads. Approximately 12±1 millions cells were harvested from the 3rd passage. The cells were round with large euchromatic nucleus and several nucleoli and small vacuoles. The cells derived from passages 1 to 4, which were grown up then, in alginate beads, showed higher staining with alcian blue. The harvested cells in some patients were immediately and successfully used for autologus transplantation. This later work will be reported separately.