فصلنامه خون
سال دوازدهم شماره 2 (پیاپی 48، تابستان 1394)

Recently the regenerative stem cell-based therapy has held great promise in treating severe degenerative diseases. SOX2 gene is highly conserved among species and plays an important role in maintenance of self renewal capacity in stem cells. SOX2 concomitant with OCT4، cMYC، and KLF4 is recruited to reprogram somatic cells towards stem cells. Today SOX2 is frequently being used in induced pluripotent stem cells generation and desired cell based therapies. The aim of this study was to clone SOX2 gene in lentiviral vector and evaluate HEK293T transduction. In the present experimental study، the coding sequence of human SOX2 gene was synthesized and received in pUC57 cloning vector. The synthesized cDNA was sub-cloned into pLEX- MCS Lentiviral vector. Lentiviral particles were produced in HEK293T cells and subsequently the HEK293T were transducted and infected cells were selected by their resistance to puromycin in the culture. Expression of SOX2 was evaluated in infected HEK293 cells by using Real-Time PCR. Constructs expressing SOX2 gene were produced and confirmed. HEK293T was successfully transducted by lentiviral particles and after 24 hours more than 90% of HEK293T represented the expression of GFP. Real-Time PCR confirmed SOX2 expression in infected HEK293 cells. HEK293T wase transducted and expressed GFP and SOX2 successfully.

Mesenchymal stem cells (MSCs) are attractive cells for cell therapy. However, the efficacy of MSCs is limited because of low survival rate following translation. In this study, MSCs were preconditioned with sub-lethal doses of H2O2. The therapeutic potential of preconditioning MSCs was examined on acute liver failure in mice. In the present experimental study, MSCs were isolated from bone marrow aspirate. Cells from passage four were treated with different concentrations of H2O2 for 24 hrs, and then were recovered in fresh medium for 7 hrs followed by exposure to lethal doses of H2O2. Cell viability was measured with WST-1 assay. In the in vivo phase, acute liver failure was induced by CCl4; then, the regenerative potential of preconditioned-MSCs was evaluated using biochemical as well as histological methods. Survival rates were higher in the engrafted mice with preconditioned-MSCs in comparison to those who received normal MSCs. Three days after cell therapy liver enzymes reached the normal level in engrafted group with preconditioned-MSCs. Interestingly, histological results revealed a significant improvement in liver regeneration potential in transplanted groups with preconditioned-MSCs. Preconditioning of MSCs with H2O2 not only enhances their survival but also increases the efficacy of MSC-based cell therapy in liver acute failure.

Acute lymphoblastic leukemia (ALL) is a neoplastic disorder and a common malignancy in children. Apoptosis is a normal physiological process which occurs during the maintenance of tissue hemostasis. The defect in this process leads to the development of cancer. It can be induced by a variety of treatments, such as cytotoxic chemotherapy. Hesperetin, a flavonoid isolated from the red fruits, has been examined with regard to the inhibition of proliferation and induction of apoptosis in pancreatic cells. Hesperetin also activates NOTCH-1 and tumor suppressor in carcinoid tumor. In this fundamental-applied research, the cell lines of C121 (Jurkat cell) were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and Penicilin/streptomycin. The growth and survival of cells with various concentrations of hesperetin was evaluated by MTS kit. The amount of cell death by trypan blue staining and flow cytometry as well as by apoptosis kit (Partec) was examined. Lymphoblastic cell lines (C121) in exposure to different concentrations of hesperetin were affected and ΜM 200 drug concentration at 48 hours was reported as inhibitory concentration or IC50. The mode of cell death induced by hesperetin was found to be apoptosis, as judged by the morphologic alteration of the cells and by Anexin v conjugated with FITC and flow cytometric analysis. Since hesperetin can induce apoptosis and reduce cell activity, it seems appropriate to be able to inhibit the growth of tumor cells.

Mesenchymal stem cells (MSCs) are the key elements of bone marrow and facilitate HSC maintenance in an invitro co-culture system through the secretion of soluble factors and cell-cell contact. Cell-derived microvesicles (MVs) or microparticles have been described as a new mechanism of cell to cell communication. In this experimental study, we obtained three placenta tissues from mothers with the informed consent under sterile condition. MSCs were isolated from the placentas; after several passages, MVs were obtained from MSC-culture-conditioned media by ultracentrifugation. Thje MVs concentration was determined in two samples by Bradford method; then, we characterized them by Transmission Electron microscopy and flowcytometer. At day 14 of the isolation, placenta-MSCs were passaged with more time for their growth required compared to BM-MSC. A different size range of microvesicles, within 40 to 160nm, was observed. The mean concentration was 125µg/mL (minimum 100µg/mL and maximum 150µg/mL) and they expressed CD29, CD44, CD73, and CD105 on their surface such as BM-MSC derived microvesicles. Microvesicles express mesenchymal cell markers that are required for adhesion to other cells. So they can contribute to the effects of mesenchymal stem cells in various cultures such as co-culture with hematopoetic stem cells.

The isolation of mesenchymal stem cells (MSCs) from the Wharton jelly (WJ) without degradation of cellular surface receptors and with the protection of cellular function, proliferation, and viability is important for research and clinical applications. In this study we have established a simple and rapid protocol without enzymatic treatment taking less time for MSCs from WJ to be isolated. Human umbilical cords were collected after full-term deliveries and transported to the laboratory in sterile phosphate-buffered saline (PBS). After the removal of cords vessels, the WJ sectioned were transferred into PBS and put on a shaker for 2 hours. The cord segments were discarded and the suspension was centrifuged and cultured in DMEM-LG supplemented with 10% FBS for 5 days. The plastic adherent cells were investigated for surface markers. Adipogenic, osteogenic, and chondrogenic differentiations were performed to investigate the mesenchymal nature. After 5 days, purified populations of spindle-shape mesenchymal stem cell appeared and the cells were then expanded until they reached subconfluence. The cell population doubling time at third (31 ± 6h) and fourth (58 ± 15h) passages was lower than other passages. The expanded cells were positive for CD 90, CD105, CD73, and CD44, and negative for CD34/45, CD133, and HLA-DR surface markers. WJ-MSCs showed the potential of the multilineage cell differentiation into adipogenic, osteogenic, and chondrogenic phenotypes. This study indicates that this non-enzymatic protocol can result in the efficient isolation of MSCs from Wharton jelly with less time taken. The expanded cells expressed characteristic markers and presented typical functional properties of MSCs such as the differentiation capacities.

During storage, functional activity of platelets is affected by platelet storage lesion (PSL). The over-expression of P-Selectin is one of the most important evidence of platelet activation, thereby monitoring P-Sel expression and shedding can be considered as an index for performance in platelet concentrates. Ten PRP-platelet concentrates obtained from Tehran Regional Educational Blood Transfusion Center were subdivided into two groups of usual platelets and leuko-reduced platelets following pre-storage filtration. Flow-cytometery and ELISA were used to evaluate P-Sel expression and shedding, respectively. Data were analyzed by one way-Anova and Mann-Whitney U tests using Graphpad software. In filtered products, P-Sel expression on days 1, 3 and 5 after storage were 13.5 ± 1.8%, 15.2 ± 1.2% and 27.4 ± 2.5%, respectively. The results showed a sinificant increase of expression in day 5, compared to that in day 1 and 3. In non-filtered products, P-Sel expression on days 1, 3 and 5 were 7.4 ± 1.7%, 21 ± 2%, and 10 ± 2.2%, respectively. Increased expression in day 3 was followed by a significant decrease in day 5, that might be due to the loss of receptors via shedding and/or microparticulation during platelet post-activation. The levels of soluble P-Sel in filtered products were significantly less than unfiltered products in day 3. Considering the expression and shedding of P-Sel, this study proposes that the leuko-reduction process favor less PSL effect during the storage of platelet products.

Thrombocytopenia is one of the most prevalent complications in ICU admitted patients that could increase the risk of hemorrhage, duration of ICU inhabitant, morbidity and mortality. Platelet transfusion is used to prevent or treat hemorrhagic conditions. Indications, triggers and response to platelet transfusion in ICU of two hospitals in Tehran were studied. In the descriptive study, 380 thrombocytopenic ICU admitted adult patients were enrolled. Indications, triggers, response to platelet transfusion (CCI), type of component, prophylactic or therapeutic transfusion and number of transfused units were studied. Among 380 patients, 124 (32.2%) received platelet transfusion, 64 (51.6%) were prophylactic and the rest were therapeutic; 30 (24.2%) used the aphaeresis platelet and the rest were administered RDP homologous PC. The mean rate of transfused units was found as 4.5 and in 58 (46.8%) of cases the non-immunologic causes of refractoriness were found. Triggers of prophylactic platelet transfusion (33 ± 4 × 109/L) and therapeutic platelet transfusion (58 ± 8 × 109/L) showed significant differences (p value = 0.04). Triggers of transfusion aphaeresis (19 × 109/L) compared to RDP homologous PC (55 × 109/L) were also shown to be significantly different (p = 0.01). The mean rates of 1st hour and 20th hour CCIs were 3700 ± 1850 and 3300 ± 1840, respectively. Almost half of transfusions in ICU were prophylactic. More than half of ICU patients who received platelet transfusion were unable to build a successful increment after a single transfusion.

The recomwell Yesinia uses YOPs (Yesinia outer membrane proteins) produced by recombinant techniques that are highly specific for the genus Yersinia. The aim of this study is to determine the prevalence of Yesinia Yop-specific IgA antibodies in 492 healthy blood donors and make comparison between seropositivity and the presence of y.enterocolitica infection in blood donors. In the descriptive study, sera from 492 healthy Iranian blood donors were studied for Yesinia Yop-specific IgA antibodies by two different techniques, enzyme immunoassay (EIA) and western blot to determine the prevalence of Yersinia antibody. Secondly, we cultured the whole blood of Yesinia seropositive donors which has been stored in 1-6 ◦ C for 35 days. Thirdly, we compared the rate of seropositivity with the results of donors’ blood cultures. The prevalence rates of Yesinia Yop-specific IgA antibodies in Tehran healthy blood donors were 12.5% and 8.5% by EIA and Western Blot, respectively. The blood cultures of all seropositive donors were negative. Although the prevalence rate of Yesinia Yop-specific IgA antibodies in Iranian healthy blood donors was high there was no correlation whatsoever between seropositivity and donors’ bacteremia.

The accumulation of white particulate matter in blood bags, with the mechanism of formation unknown, has been rarely reported. However, the high number of platelets, blood lipids, and the like could help the white matter form. Case: A 450 mL blood bag coated with 63 mL CPDA1 including the white matter detected was transferred from the blood center to the central quality control department. All test results on RBCs, white cells, platelets, hemoglobin, hematocrite, protein sera, triglyceride, and cholestrol were negative. The donor had no record of any medicine consumption. The microbial culture of white matter was negative. Although no correlation has been clearly found between the accumulation of white matter and transfusion reactions, it is recommended to prevent administering the white matter including blood bags to the patient; these blood bags should be quarantined for further analysis. The appearance of the blood bags before transfusion should be checked.

Umbilical cord blood (UCB) is an accessible source of hematopoietic stem cells (HSCs). Along with the advantages, UCB also has limitations: the low volume and the absolute number of HSCs available in UCB leading to the delayed engraftment. Given the limitations, many investigators have sought to accelerate engraftment and increase the absolute number of stem cells in UCB units. In the present study more than 200 published articles about UCB were reviewed. This review article is aimed to focus on the importance of using cord blood, the nature of stem cells in cord blood, and the ex vivo expansion techniques of UCB HSC. UCB HSCs possess higher proliferative potentials and contain a higher proportion of primitive compartment as compared to bone marrow and peripheral blood. Several studies have reported the presence of different cell populations besides HSCs in cord blood that enable the use of these sources in immunotherapy, tissue engineering, and regenerative medicine. Thus, the strategies to isolate and expand selected subpopulations from UCB and the use of these cells in treatment of various diseases are the areas of active research. Umbilical cord blood is an attractive source in both research and modern clinical applications providinh a potentially useful alternative for patients who do not have an HLA-matched bone marrow donor. Besides the safety and feasibility of UCB, the other areas including the acceleration of the engraftment, the extension of access, the quality assurance, and the outcomes in the specific subgroups of patients are also required to be investigated.