Cell Journal (Yakhteh)
Year:2007 | Volume:9 | Issue:2 (34)|Papers Count:8

Objective: Dendritic cells (DCs) are the most effective antigen-presenting cells with many applications. However, DCs can be generated in vitro by various methods from bone marrow cells, each with varying pros and cons. Thus, we evaluated three different methods to generate DCs from mice bone marrow cells.Materials and Methods: BM cells from C57BL/6 mice were cultured in the presence of 1000U/ml of GM-CSF either with or without 500 U/ml of IL-4 for 6 days as first and second method respectively. In the third method, BM cells were cultured in bacterial plates in the presence of 200 U/ml GM-CSF for 10 days. For further maturation, the cultures were extended for two more days using 50ng/ml TNF-a. The purity of the obtained DCs, their subtypes and maturation states were determined using flow cytometric analysis and the capacity to induce the allogenic T cell proliferation was determined using [3H]-thymidine incorporation.Results: The purity of generated DCs (CD11c+) in both methods of GM-CSF plus IL-4 and culturing in bacterial plates was more than that of GM-CSF method. Culturing in bacterial plates resulted in the generation of higher number of DCs. However, DCs were resistant to maturation induction by maturation factors. Expression of maturation markers (CD86 and MHC class II) on DCs was up-regulated in the presence of IL-4 compared to the other two methods and the cells acted more efficiently in the induction of allogenic T cells proliferation. All the three methods resulted in the successful generation of myeloid (CD11b+) DCs.Conclusion: A combination of GM-CSF and IL-4 results in the generation of more pure, more mature, and functionally more effective DCs.

Introduction: The present study examines the effects of increased heat shock proteins expression in MDA-MB468 adeno-carcinoma cells on expansion and differentiation of hematopoetic stem cells from human umbilical cord blood.Materials and Methods: Highly purified human CD34+ cells (Magnetic bead isolation) derived from umbilical cord blood were cultured, under three conditions, in the presence of FLt3L, SCF, GMCSF, IL-3, TPO and MDA-MB 468 Carcinoma lysate (MDA); heat treated MDA-MB468 lysate (MDA-HSP) or without tumor lysate as control group. Flow cytometry and direct scoring of solid cultures were done to evaluate the proliferation, and colony forming ability and surface markers of CD34+ Cells on days 0, 7 and 14 of culture.Results: Long term culture of CD34+ Cells in the presence of tumor lysates caused their differentiation with decrease in cell expansion and colony forming unit potency. Phenotype of cultured cells was changed toward myeloid or lymphoid lineages. Comparison of the three groups showed that, MDA caused the lowest proliferation and CFC followed MDA-HSP. Among the groups, MDA and MDA-HSP, Stimulated the differentiation toward lymphoid cells Specially B Cells (CD19+), NK cells (CD56+) and myeloid progenitor cells (CD14+). However, CD14+ increased in control group.Conclusion: Tumor cells contain substances regulating development and decreasing proliferation and colony forming potential of HSCs. Heat treated tumor cells contain HSPs and other substances preserving some expansion and colony forming properties of hematopoietic stem cells. It could be argued that these substances may cause differentiation of HSCs into immune cells such as NK and myeloid cells that induce strong anti tumor immune responses.

Objective: The objective of this study was to evaluate the relationship between spermiogenic defects such as protamine deficiency, and acrosomal integrity with fertilization and pregnancy rate in IVF patients.Materials and Methods: Density, motility and morpology of semen samples from 70 infertile couples undergoing IVF at Isfahan Fertility and Infertility center were analyzed according to WHO criteria. Protamine deficiency and acrosin activity were assessed by Chromomycin A3 (CMA3) and Gelatinolysis test. Statistical analysis was carried out using the Statistical Package for the Social Studies (SPSS 11.5) and P-value lower than 0.05 was considered to be significant.Results: CMA3 positivity, percentage halo formation, means halo diameter and sperm morphology showed a significant correlation with fertilization rate. The rate of halo formation and mean halo diameter showed a significant correlation with CMA3 positivity. None of the parameters were significantly different between pregnant and non - pregnant patients. However, the rate of halo formation showed a closely significant difference (r=0.306; P=0.058).Conclusion: The results of the present study indicate that sperm acrosomal integrity assessed by percentage halo formation has profound effects on pregnancy outcome in IVF procedure.

Objective: Endothelial cells are subjected to mechanical forces caused by pulsatile flow and pressure leading to hemodynamic shear stress and circumferential stress on endothelial layer. The study aimed at a quantitative evaluation of endothelial cell morphology by application of cyclic loading. Endothelial cells respond to applied loads with adaptation and remodeling. Such mechanisms are major determinants of the function of cardiovascular system.Materials and Methods: A cyclic loading test device for cellular engineering was designed and manufactured to operate in wide ranges of applied strains, load frequencies and amplitudes, and stretch to release ratio. Human umbilical vein endothelial cells were cultured on a medical grade silicon membrane and the membrane was stretched inside an incubator with strain amplitudes of 10%-25% and test durations of 4-10 hours. Quantitative cellular morphological changes by cyclic loading were analyzed using designed image processing software.Results: The results indicated marked changes in orientation of cells, from a random orientation to 70-80 degree alignments after loading. Endothelial cells elongated due to loading and the shape index (SI) decreased significantly.Conclusion: It was concluded that morphology of endothelial cells is altered by cyclic loading: load amplitude and number of load cycles. The results may have applications in pathology of endothelial cells due to hypertension and in vascular remodeling following the effects of different loads on endothelial cells.

Objective: Cloning an individual by transferring somatic nuclei into enucleated recipient oocytes has already been well established. This technology also offers new opportunities to restore threatened species with interspecies somatic cell nuclear transfer (SCNT). However, few mammalian species have been studied for their reproductive biology whereas huge differences have been observed between these species. This study evaluated the similarities and genetical relationship of germ lines and the reproductive biology between domestic (Ovis aries) and threatened wild sheep (Ovis orientalis isphahanica).Materials and Methods: Six populations of wild and domestic sheep were sampled and analyzed for chromosome number, interbreeding capability and fecundity. Resulted hybrids (male or female) were investigated for survival, karyotyping and fertility.Results: Both the domestic and wild sheep uniformly exhibited a 2n of 54 and were able to crossbreed and induce sustainable pregnancy into the counterpart species. The resultant hybrids (male or female) which were produced by either wild ram × domestic ewe or domestic ram × wild ewe had identical chromosome number (2n=54). Normal genital apparatuses and fertile conditions were observed in both types of adult hybrids.Conclusion: The results indicated a very close generational relationship between the wild and domestic sheep species and also the possibility of domestic species to be used as an abundant genetic background for saving endangered wild sheep via SCNT.

Objective: Effects of immediate TNF-a exposure on phenotype and function of dendritic cells (DCs) derived from cord blood mono nuclear cells.Materials and Methods: Umbilical cord blood MNCs were isolated from healthy mothers and were divided into TNF (+) and TNF (-) groups. Both were cultured using SCF, Flt3L, GM-CSF and IL-4. But, three ng/ml of TNF-a was first added in the culture of TNF (+) group. All cells were cultured for 14 days and matured with TNF-a or LPS for additional four days. Light microscopic and flowcytometric analyses were performed on days 0, 7, and 14 of both cultures. MLR and cytokines assays were used to characterize the function of immature and mature DCs.Results: Co-culture of cord blood monocytes and hematopoietic stem cells led to the production of DCs with a characteristic veiled appearance and were consistent with a DC panel of surface markers. However, immediate exposure to TNF-a enhanced the survival of culturing cells in the first week of culture and produced mature DCs with higher maturation markers and IL-12 production. Addition of TNF-a as a maturation marker led to the production of matured DCs and also certain immature and hematopoietic stem cells with higher level of IL-10 production.Conclusion: This study developed a simple, easy and cost effective way to generate DCs from non fractionating mononuclear cells. It seems that primitive DCs and monocytes in the MNCs are contented in the presence of TNF. This will lead different hematopoitic stem cells to myeloid pathway and results in DCs.

Objective: The spermatogonial stem cells (SSCs) form the basis of spermatogenesis. In vitro culture of germ cells is useful for the purification and increasing of SSCs for transplantation. In the present study, we examined: in vitro the effects of co-culture with sertoli cells, GDNF, SCF and GM-CSF on efficiency of spermatogonial cells colony formation and spermatogenesis induction in the recipient testes after transplantation with SSCs in the colonies.Materials and Methods: Both Sertoli and spermatogonial cells were isolated from adult mouse testes by enzymatic digestion. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against oct-4 and vimentin; and also transplantation of the cells. Isolated spermatogonial cells were treated either with various concentrations of GDNF (1, 40, 100ng/ml), SCF (1, 40, 100ng/ml) and GM-CSF (0.1, 0.1, 1ng/ml) or with co-culture with Sertoli cells during three weeks. Colony assay was done using a light microscopy. For transplantation, spermatogonial cells of the colonies were transplanted into the seminiferous tubules mouse which was irradiated with 14Gy at 10 weeks of age, via rete testis. The statistical significance between mean values was determined using repeated ANOVA test. p<0.05 were considered to be significant.Results: Results indicated that GDNF is the best factor for in vitro colonization of adult mice spermatogonial cells compared with the other cytokines and growth factors. Co-culture system with Sertoli cells was chosen as colonizer. Because co culture showed a significant increase in number (25.1±5.2) and diameter (205.8±50.1µm) of colonies compared with growth factors in both treated and control groups. Spermatogonial stem cells in the colonies may induce spermatogenesis in the recipient testes after transplantation.Conclusion: Co-culture system with Sertoli cells increases adult spermatogonial cell colony formation in vitro.

Objective: Proteolytic enzymes, especially collagenase, are used to digest extracellular matrix, cells isolation and primary culture. It is important to find new sources of plant or animal protease instead of bacterial or tissue collagenase. In the present research, actinidin, a plentiful protease in kiwifriut (Actinidia deliciosa), was used to isolate human umbilical vein endothelial cells.Materials and Methods: Human umbilical vein endothelial cells (HUVEC) were isolated using different doses of actinidin (from 1 to 16mg/ml) and different incubation times (from 10 to 60 minutes). Freshly isolated endothelial cells were cultured in MCDB131 medium. Endothelial cells were identified by their non-overlapping cobblestone morphology and immunostaining. The viability of separated cells was assessed by trypan blue exclusion test.Results: Actinidin in concentration of 4mg/ml for 20-30 minutes selectively isolates human umbilical vein endothelial cells with minimal contamination of other cell populations. The viability of separated cells was estimated 90-95% in this situation.Conclusion: The results showed that actinidin has not toxic effect on separated cells and is a novel and suitable protease for isolation of HUVEC cells.