مهین نیکوگفتار ظریف

Proliferation and differentiation of mesenchymal stem cells (MSCs) in culture media requires the presence of nutrient supplements such as bovine fetal serum (FBS), which increases the risk of prion/xenotic infections. Platelet lysates as a rich source of growth factors and cytokines can replace FBS in cell culture medium. This study aimed to compare the effect of platelet lysates derived from cord blood and peripheral blood on the proliferation and differentiation of MSCs.

In this experimental study, human MSCs were isolated from placental tissue and identified by flow-cytometry. 3 Three platelet bags were collected and platelet lysates were prepared by repeated freezing/thawing method. MSCs were cultured in different media and evaluated for their differentiation capacity, cultured in osteogenic and adipogenic differentiation media with Alizarin Red and Oil Red-O staining. TGF-β levels were assessed by ELISA. Statistical analysis was performed using SPSS22 software and One-Way ANOVA statistical test.

The proliferation rate of MSCs in media containing Cord/Peripheral Blood Lysate (CBL or PBL) was not significantly different from that of media containing 5% FBS. Also, the rate of proliferation in the medium containing CBL plus cord blood serum was not significantly different from the medium containing 10% FBS. Also, the ability of MSCs’ differentiation into osteocytes and adipocytes in media containing CBL or PBL was well preserved.

Platelet lysates can be a suitable alternative to animal serum in cell culture medium. In addition to the growth and proliferation of MSCs, these factors maintain the ability to differentiate into osteocytes and adipocytes in environments containing these factors.

Safe and effective gene therapy is considered as one of the therapeutic goals in many diseases. Due to the important role of stem cells in cell therapy, this study aimed to produce human adipose-derived mesenchymal stem cells (hASCs) using the miR-34a overexpression.

The hsa-mir-34a precursor sequence was cloned into the PCDH lentiviral vector. The recombinant vector and two helper vectors, i.e. psPAX and pMD2, were transferred into HEK-293T cell line by calcium phosphate method. Viral supernatant was collected and concentrated by ultracentrifuge. On the fourth day, transduced HEK-293 T cells were analyzed by flowcytometry. After the determination of viral concentration, hASC cells were transduced with condensed viruses. RNA extraction and cDNA synthesis were performed in order to assess miR-34a expression level by Real Time PCR.

The hsa-mir-34a precursor sequence cloned into PCDH vector was confirmed by colony-PCR and DNA sequencing. Transduction of HEK-293T cells and hASCs were confirmed under fluorescent inverted microscope and flowcytometry. The assessment of miR-34a expression level in infected cells with recombinant virus showed that the expression ratio of miR-34a in the test group was significantly higher than the control group (P=0.001).

This study showed that lentiviral systems can be used to insert actopic genes like miR-34a into cells. It also showed that genetically manipulated stem cells can be used as a delivery system to deliver miR-34a or other genes.

Platelets undergo a series of biochemical, functional, and morphological changes during storage that are collectively called platelet storage damage, which can reduce platelet function and survival. Oxidative damage is one of the leading causes of platelet storage damage. Therefore, in the present study, an attempt has been made to determine the effect of oxidative stress on platelets during storage. This research was conducted in 2017 at the Research Center of the Iranian Blood Transfusion Organization.

In the current experimental study, 10 platelet concentrate bags prepared by platelet-rich plasma (PRP) were examined. Research parameters include measurement of mitochondrail reactive oxygen species (ROS), concentration of malondialdehyde (MDA), concentration of nitric oxide metabolites (nitrate / nitrite), activity of lactate dehydrogenase enzyme (LDH) and platelet count on days 1 ,3 ,5 and 7 of platelet storage. Statistical analysis of variance was used to describe the obtained data

According to the results, ROS (16.22±37), MDA (11.6±22.2) concentration and LDH enzyme activity (310±3680) increased significantly (P<0.001) while PLT count was significantly decreased (P<0.001).

It seems that oxidative damage occurred in platelet with increasing radical oxygen species and lipid peroxidation which can reduce platelet count and platelet viability during platelet storage. Using antioxidant as an additive in platelet concentrate may decrease oxidative damage and increase platelet viability and quality during storage.

 The genome of cell lines is nowadays edited to create disease models and treat them. Of course, the size of the cas9 gene has caused problems like the low efficiency of the CRISPR system. To solve this problem, Cas9 expressing cell lines have been generated in which CRISPR RNA should only be transfected to the cell.   This article is experimental. PGK-PURO / CMV (PPC) fragment was amplified with PCR from pAAVS1-puro-DNR vector and cloned in pTG19-T vector. The PPC fragment from this vector was removed by KpnI and EcoRI enzymes. Also the pCas-Guide-AAVS1 vector was subjected to the same enzymatic cutting and its attachment to the PPC fragment resulted in the production of the pPPC-Cas vector. After optimizing the electroporation conditions, the pPPC-Cas vector was electroporated into K562 cells and puromycin -resistant cells were selected and Cas9 expression level was evaluated by Real-time PCR.   A PCR fragment of 2514 bp was amplified. The vector pPPC-Cas was cloned in two steps. puromycin -resistant transfected cells were selected. Clonal selection was carried out and three colonies with high, medium and low expression level of Cas9 were isolated.     The Cas9-expressing K562 cells derived in this study can be applied both for functional genomic researches and design cellular models of human diseases in future.

Background and Objectives The low number of umbilical cord blood cells is an important barrier to successful bone marrow transplantation. Therefore, the growth of these cells while maintaining their functional characteristics is of great importance. Culturing cells on 3D scaffolds like DBM has greatly contributed to the simulation of the mechanical and chemical micro-environment of the bone marrow tissue. There are also specific compounds for proliferation without differentiation of cells one of which is nicotinamide.
Materials and Methods In this experimental study, hematopoietic stem cells (HSCs) were cultured 7 days in DBM under the following conditions of (1) negative control, (2) cytokine, (3) nicotinamide, (4) nicotinamide and cytokine. After 7 days, cell count, purity using flow cytometry, colony forming unit assay and apoptosis were evaluated and performed. The data were analyzed using Anova.
Results Our results indicated that in all cases, the combination of nicotinamide and cytokine was accompanied with more proliferation, more CD34 cells, less apoptosis, and higher colony-forming ability than other groups.
Conclusions Nicotinamide is a very suitable compound for the development of umbilical cord blood cells given the role of the former in preventing epigenetic changes that are the outcome of laboratorial cell culture and the simulation of bone marrow niche by DMB.

The main method of separating platelet microparticles (PMP) is based on the centrifugation speed and time. Due to the high cost of determining the number of PMP via micro-particles (micro bead) and also the necessity of using an expensive device such as a flow cytometer, it seems that Bradford method would be rather an inexpensive, fast and efficient way to determine the concentration of PMP. Therefore, in this study the effect of different factors, such as speed and time of centrifugation and time of freezing on the concentration of PMP in the platelet concentrates bags was studied.
We studied two different speeds of centrifugation for separating PRP. In the first protocol for preparation of PRP, the platelet bags were centrifuged at 1500g for 15min and in the second protocol; they were centrifuged at 5000g for the same duration. To evaluate the effect of time, microparticles were separated in 16000g for 20 and 2 min. To determine the concentration of PMP, Bradford method was used. To evaluation the effect of freezing, the PRP was prepared at 300g for 20 min, and then it was freezed in -80˚c for five days. Flow cytometery analysis was performed for microparticles identification.
PMP concentrates with the 1500g centrifugation speed showed higher concentration (P The result of this study showed that the reduction of centrifugation speed could produce higher levels of the microparticles. In addition, the time of separation in the final stage had no significant effect on PMP isolation. Freezing could lead to higher PMP concentration.

Cord blood CD133+ cells are able to maintain long-term hematopoiesis and to differentiate to different hematopoietic lineages. CXCR4 overexpression is involved in successful transplantation of hematopoietic stem cells in the bone marrow. Platelet micro particles (PMP) contain CXCR4 markers and are able to transfer them into hematopoietic stem cells. Therefore, considering the importance of CD133+ cells as primitive HSCs, the effect of platelet micro particles on the expression levels of CXCR4 and CD34 markers in these cells was examined. this experimental study, cord blood CD133+ cells were isolated by MACS. Isolated cells were cultured into three groups one as control without adding PMP and the other two groups with PMP with concentrations of 5 and 10 μg/mL. The cells were cultured for five days in stem span medium. Expression of CXCR4 surface marker was analyzed by flow cytometery; the total cell number was counted by hemocytometer and colony forming units (CFU) were measured by colony assay. Fold increase of CD34+ and CXCR4+ cells in the control group and in the presence of 5 and 10 µ g/mL of PMP were 52.6 ± 4.16, 64.2 ± 6.91, and 67.2 ± 6.76, respectively (p= 0.047). CXCR4+ cell percentage in the presence of 10 µ g/mL PMP compared to control cells (63.8 ± 6.4) was 72.8 ± 4 (p= 0.049). Exposure of CD133+ cells isolated from cord blood to PMP with 10 μg/mL concentration increased the expression of CXCR4 surface marker significantly.

Cord blood CD133+ stem cells are highly proliferative primitive hematopoietic progenitors. These cells can also differentiate to other cells especially neural cells. The PAX6 gene belongs to a family of genes that play a critical role in the formation of tissues and organs during embryonic development. The PAX6 protein is thought to activate genes involved in the formation of the eyes, brain and spinal cord (central nervous system), and the pancreas. The purpose of this study was to assess PAX6 (5a) gene transfer in cord blood CD133+ stem cells and evaluate its effect on differentiation. In this experimental study, cord blood stem cells were collected and mononuclear cells isolated by ficoll; then, CD133+ cells were obtained using the CD133 MicroBead Kit in combination with the autoMACS Separator. These cells were cultured in Stem Span media. Next, HEK293T packaging cells were co-transfected with PLEX-MCS, PsPAX2, and pMD2G by calcium phosphate method. Lentiviral vectors were collected and concentrated. The appropriate amount of viruses was used to infect CD133+ cells. The successful transduced cells were selected by puromycin resistance. After two weeks, the expression of Rhodopsin, CHX10, Thy1, Nestin, and PAX6 proteins was assessed by immunocytochemisrty method. Two weeks after infection, the expression of Rhodopsin, CHX10, Thy1, Nestin, and PAX6 proteins were detected in treatment cells and the expression of Rhodopsin and Nestin in control cells. The results showed CD133+ cells differentiated into progenitor neural like cells and retinal neural like cells including ganglion like cells.

Mesenchymal stem cells are adult, non-hematopoietic, mulipotent and self-renewal cells. These cells were first isolated from bone marrow and considered as the most important cells in cell therapy. These cells have been used in clinical trials (animal models) about more than a decade. Regarding immunomodulatory properties through immunosuppressive factors and their repair potency, these cells have been widely used in the treatment of immunological (chronic and autoimmune diseases) and non-immunological diseases (tissue engineering, tissue repair, degenerative and malignant diseases.(This review aimed to study the therapeutic roles, immunobiology and challenges ahead of mesenchymal stem cells. At first articles related to mesenchymal stem cells were searched from valid databases such as Wily Online Library, ISI web of science, Springer Link, Sciencedirect, Pubmed, google scholar, SID, and ISC. Then, the related articles studied from 2002 to 2014 were searched. They were about the modulatory role, therapeutic effects (in immunologic and non-immunologic diseases) and clinical applications of mesenchymal stem cells. The obtained results showed a high potential of mesenchymal stem cells, isolated from different sources in clinical trials (preclinical and clinical). Mesenchymal stem cells have a key role in immunomodulation through secreted factors and cell-cell contact in inflammatory diseases, as well as tissue restoration by homing via adhesion molecules and chemokines in cells that has been destructed due to physiologic or pathologic causes (non-inflammatory and destructive diseases). In addition, it has anticancer features for nanoparticle delivery to tumors and consequently angiogenesis prevention. Despite mentioned points, use of mesenchymal stem cells has some risks such as tumor induction, infection transmission, and ectopic tissue formation in receiving individual. The therapeutic role of mesenchymal stem cells in destructive and autoimmune diseases has been proved. Given challenges in using mesenchymal stem cells in cell therapy and the standards (according to protocols), these cells can be used widely in the future.

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.

Different processing methods are being used to improve the quality of hematopoietic stem cell transplantation. Using hydroxyethyl starch, simple centrifugation and Sepax automation, this study was aimed to compare these three conventional methods. 90 cord blood samples were taken and processed by hydroxyethyl starch, simple centrifugation and Sepax automation methods. Then they were subjected to total nucleated cell (TNC) counting and CD34 positive counting as well as colony assay. Finally, all data were analyzed using one-way analysis of variance (ANOVA) and ps less than 0.05 were considered statistically significant. The TNC recoveries in hydroxyethyl starch, simple centrifugation and Sepax automation methods were 76%, 71% and 80%, respectively (p> 0.05). The CD34+ cell recoveries in the Sepax automation and in the other two methods were 91% and 85%, respectively (p> 0.05). Also, the colony assay recoveries were not significantly different among the three methods (p> 0.05). No significant difference was seen in TNC number, CD34 positive counting and colony formation among the three different methods.

Hematopoietic stem cell transplants are routinely used to treat patients with cancers and other disorders of blood and immune systems. Osteoblasts constitute part of the stromal cell support system in marrow for hematopoiesis by participating in the formation of the HSC niche. It is believed that interaction between hematopoietic cells and bone forming osteoblasts regulate each other’s function. It is established that acute blood loss in animal models activates bone formation and niche development because of EPO stimulation. In this experimental study we have examined the co-culture of HSCs derived from cord blood which treated with EPO in vitro, on osteoblastic differentiation of mesenchymal stem cells. In this experimental study MSCs isolated from bone marrow and co-cultured with CD 34+ CD38- HSCs isolated from cord blood. These co-cultured cells were treated with different doses of erythropoietin for 14 days, after that RNA were extracted from MSCs and analysed with RT-PCR to evaluate the expression of osteopontin and osteocalcin. Alizarin red and alkaline phosphatase staining were done for osteoblastic differentiation. Osteopontin and osteocalcin were expressed in MSCs. Cellular staining were positive for osteoblastic differentiation. Differentiated cells expressed osteoblastic markers. These data suggest that EPO regulates the osteoblastic differentiation from bone marrow MSCs in vitro.

The low frequency of hematopoietic stem cells in umbilical cord blood is the main factor of its application to be restricted for transplantation. If hematopoietic and mesenchymal stem cells of placenta can be isolated and transplanted with cord blood cells, the problem of cell dose limitation will be solved due to increase in the number of primary hematopoietic stem cells and homing of cells through mesenchymal stem cells. Thus in this study, we tried to isolate the maximum number of hematopoietic and mesenchymal stem cells from placenta by enzymatic methods and evaluate their properties. Five different placenta tissues were treated with collagenase and their red blood cells were omitted with the lysis buffer; then, the remaining cells were studied for the presence of hematopoietic and mesenchymal stem cells. The result showed that 6.02 ± 0.6 and 4.78 ± 0.8 percent of isolated cells were CD34+ and CD34+ together with CD38-, respectively. These cells having generated all hematopoietic lineages and adherent stromal cells with the property of mesenchymal stem cells were obtained from the culture of placenta cells. The presence of a remarkable number of CD34+ cells and mesenchymal stem cells in placenta tissue showed that transplantation of these cells with umbilical cord blood cells can increase the quality of transplantation.

Mesenchymal stem cells are fibroblasts with the capacity of proliferation، colonogenesis، and differentiation to the mesodermal cells that are known as the source for cell therapy purposes. These cells are present in some adult and embryonic tissues in which the bone marrow serves as the main source of human mesenchymal stem cells. Because of some limitations in using the bone marrow، in this study we established a method for isolation of mesenchymal stem cells from placenta and characterized their properties. In this experimental study، we obtained 3 placenta tissues from informed consent mothers after normal vaginal delivery. Mesenchymal stem cells were isolated from the placenta، cultured، and were then examined for their proliferation، colonogenesis، immunophenotyping and differentiation capacities from passages 3 and 7. Our results showed the high capacity of proliferation and colonogenesis of placental derived mesenchymal stem cells. Immunophenotyping confirmed more than 95% purity of isolated cells; their surface antigen expression showed the phenotypical properties like those of bone marrow derived mesenchymal stem cells. The cells had the osteocytic and adipocytic differentiation capacity. In this study we successfully isolated mesenchymal stem cells from placenta and cultured them without any alteration in their capacities; consequently، we found out that we can isolate and expand these cells as an alternative source for cell therapies.

Umbilical cord blood is a promising alternative source of hematopoietic stem/progenitor cells (HSPCs) for allogeneic hematopoietic stem cell transplantation. Although the use of UCB for transplantation has plentiful advantages in comparison with other cell sources، the low number of HSPCs led to cell delayed recovery. Therefore، ex vivo expansion of HSPC before transfusion could induce faster cell recovery. CD34 positive cells were positively enriched using MidiMACS system and cultured on aminated PS nanofibers، aligned PCLs، and random PCLs. Cell expansion and quantification of cell apoptosis were carried out in expanded cell populations in different conditions by the flowcytometric method. Microscopic and flowcytometric analysis revealed the differentiation of CD133+ cord blood hematopoietic stem cells to erythroid lineage. Flow cytometry showed that both of the PPARγ agonists were able to diminish Transferrin receptor and glycophorin A positive cell population significantly. The inhibitory effect of PPARγ agonists on erythroid differentiation was dose-dependent. The inhibitory effect of Troglitazone on colonies formation of Erythroid was markedly higher than Bicaline. This study demonstrated that aminated PS nanofibers has positive effects on the expansion of CD34+ cells compared to 2D culture system by increasing cell expansion and cell apopotosis inhibition. However، aligned and random PCL nanofibers did not show supportive effects on cell proliferation.

The correlation between hematopoietic stem cells (HSCs) and the cells comprising the niche especially osteoblast cells is critical for maintaining stem cell activities. Yet little evidence supports the concept that HSCs regulate development of the niche. If true، it may explain why many hematopoietic defects are accompanied by changes in the osseous architecture and provide new therapeutic targets for regulating bone formation. In this exprimental study، we cocultured bone marrow mesenchymal stem cells (MSCs) with umbilical cord blood HSCs in stem span media for 3 days. Then MSCs were differentiated to osteoblast cells by using osteogenesis kit. Evaluation of osteogenic differentiation of MSCs in different days came out to be: expression of osteopontin and osteocalsin (RT-PCR on days 4 and 6)، expression of CD90 (flow cytometry on day 6)، expression of alkaline phosphatase enzyme (alkaline phosphatase staining) and mineralization of MSCs (alizarin red staining on day 10). Our results showed early expression of osteopontin and osteocalsin in HSCs coculture with MSCs. These findings support the role of HSCs in induction of osteoblastic differentiation of MSCs. Decrease in the expression of CD90، higher activation in cell alkaline phosphatase enzyme، and mineralization confirmed the results. In the field of human stem cells، our ex vivo findings demonstrated that HSCs can enhance differentiation of MSCs toward osteoblast cells thereby participating in formation of their niche.