mahin nikougoftar zarif

In the current study we have aimed to find the effects of Resveratrol treatment on platelet concentrates (PCs) at the dose dependent manner. We have also attempted to find the molecular mechanism of the effects.

The PCs, have received from Iranian blood transfusion organization (IBTO). Totally 10 PCs were studied. The PCs divided into 4 groups including untreated (control) and treated by different dose of Resveratrol; 10, 30 and 50 µM. Platelet aggregation and total reactive oxygen species (ROS) levels were evaluated at day 3 of PCs storage. In silico analysis was carried out to find out the potential involved mechanisms.

The aggregation against collagen has fallen dramatically in all studied groups but at the same time, aggregation was significantly higher in the control versus treated groups (p<0.05). The inhibitory effect was dose dependent. The aggregation against Ristocetin did not significantly affect by Resveratrol treatment. The mean of total ROS significantly increased in all studied groups except those PCs treated with 10 µM of Resveratrol (P=0.9). The ROS level significantly increased with increasing Resveratrol concentration even more than control group (slope=11.6, P=0.0034). Resveratrol could potently interact with more than 15 different genes which, 10 of them enrolled in cellular regulation of the oxidative stress.

Our findings indicated that the Resveratrol affect the platelet aggregation at the dose dependent manner. Moreover, we have also found that the Resveratrol play as double-edged sword in the controlling oxidative state of the cells. Therefore, Using the optimal dose of Resveratrol is the great of importance.

Conditioned medium (CM) derived from mesenchymal stem cells (MSCs) contains bioactive molecules including microRNAs (miRs) that could be a potential tool for controlling cancer cells' behavior. Due to the properties of CM, this study assesses the effects of miR-34a related MSC-CM on tumor behavior through the evaluation of migration, invasion, apoptosis, and PDL1 expression in breast cancer cell lines. The miR-34a overexpression vector or scramble control was produced using lentiviral vectors, DNA cloning, and the transfection of the HEK-293T cell line. It was then transduced into human adipose-derived mesenchymal stem cells (hAD-MSCs). MSC-CMs were collected and added onto MDA-MB-231 cell lines. The functional evaluations were performed by transwell, wound healing, and Annexin V/PI methods on the treated MDA-MB-231 cell lines. The PDL1 expression was also assessed by Real-time PCR and western blot. The findings of this study showed that ectopic miR‑34a expression was significantly upregulated in manipulated hASC with miR-34a (p<0.0001). Treatment of MDA-MB-231 cell line with miR-34a-hAD-MSC-CM, scramble-hAD-MSC-CM, or hAD-MSC-CM displayed not only a reduction in the number of migrated or invaded cells (p=0.01) but also an increase in the apoptotic cells in the test group (p=0.02) when compared to the control groups. It also showed down-regulation in the gene (p=0.05) and protein expression levels of PDL1 in the test group. The results of the present study showed that simultaneous application of miR-34a and MSC-CM can be considered as a new method for changing the cancerous microenvironment; and therefore, as a potential strategy in breast cancer therapy.

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.

In order to clarify the role of microRNAs (miRNA) in megakaryocyte differentiation, we ran a microRNA microarray experiment to measure the expression level of 961 human miRNA in megakaryocytes differentiated from human umbilical cord blood CD133 cells.
In this experimental study, human CD133 hematopoietic stem cells were collected from three human umbilical cord blood (UCB) samples, and then differentiated to the megakaryocytic lineage and characterized
by flow cytometry, CFU-assay and ploidy analysis. Subsequently, microarray analysis was undertaken followed by quantitative polymerase chain reaction (qPCR) to validate differentially expressed miRNA identified in the microarray analysis.
A total of 10 and 14 miRNAs were upregulated (e.g. miR-1246 and miR-148-a) and down-regulated (e.g. miR- 551b and miR-10a) respectively during megakaryocyte differentiation, all of which were confirmed by qPCR. Analysis of targets of these miRNA showed that the majority of targets are transcription factors involved in megakaryopoiesis.
We conclude that miRNA play an important role in megakaryocyte differentiation and may be used as targets to change the rate of differentiation and further our understanding of the biology of megakaryocyte commitment.

Mesenchymal stem cells (MSCs), as major stem cells for cell therapy, have been studied from different aspects in preclinical and clinical settings for more than a decade. These cells modulate the immune system (humoral and cellular immune responses) in vitro by producing soluble factors (anti-inflammatory molecules) and/or making cell-cell contacts. Hence, they could be used in regenerative medicine, tissue engineering and immune therapy. MSCs-based therapy have been recently used for treatment of cancer regarding the migratory potential of these cells towards tumor cells which makes them considerable candidates, also for cell therapy in both allogeneic and autologous settings. So, this review attempts to focus on the factors secreted by MSCs such as cytokines, their functional role in mounting and controlling immune responses mediated by different immune cell subpopulations and their significance in regenerative medicine in clinical trials. Although, further studies remain to be done to increase our knowledge of regulating development mechanisms, homeostasis and tissue repair in order to provide new tools to implement the efficacy of cell therapy trials. Although MSCs have been proved safe and effective for cell therapy, there are still challenges to overcome before widely applying MSCs in clinic.

MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis.
In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, β, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14th and 21th after transduction.
Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokines.

This study was designed to evaluate platelet transfusion outcome via flow cytometric monocyte phagocytic assay (FMPA).
Fifteen patients with a history of multiple platelet transfusions and fifteen controls were enrolled in this study. CMFDA-labeled platelets were incubated with patient's sera and were finally incubated with monocytes in a tube and analyzed by flow cytometry. Monocytes that phagocytosed platelets were detected as a CMFDA-positive platelet population via monocyte gate. The FMPA results were compared with CCI results for the patients.
The FMPA result correlated with 1-hour (r = -0.885, P = 0.001) and 24-hour (r = -0.884, P = 0.001) CCI. There is a significant difference in means of FMPA results between the patients with immune platelet refractoriness (68.46 ± 10.4%), non-refractory group (37.73 ± 15.21%) and the control group (18.27 ± 2.86%).
Our data showed that FMPA has good results in evaluation of platelet transfusion outcome and may be useful as an indicator of platelet transfusion response.

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.

Hematopoietic stem cells (HSCs) transplantation using umbilical cord blood (UCB) has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand (FLT3-L) as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell (MSC) feeder. In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count (TNC), cluster of differentiation 34+ (CD34+) cell count, colony forming unit assay (CFU), long-term culture initiating cell (LTC-IC), homeobox protein B4 (HoxB4) mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines (P<0.05). FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased (P<0.05). FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture.

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.

Despite of many benefits, umbilical cord blood (UCB) hematopoietic stem cell (HSC) transplantation is associated with low number of stem cells and slow engraftment; in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte (MK) progenitor cells can shorten this period. In this study, we evaluated the cytokine conditions for maximum expansion and MK differentiation of CD133+ HSCs. In this experimental study, The CD133+ cells were separated from three cord blood samples by magnetic activated cell sorting (MACS) method, expanded in different cytokine combinations for a week and differentiated in thrombopoietin (TPO) for the second week. Differentiation was followed by the flow cytometry detection of CD41 and CD61 surface markers. Colony forming unit (CFU) assay and DNA analysis were done for colonogenic capacity and ploidy assay. CD133+ cells showed maximum expansion in the stem span medium with stem cell factor (SCF) + FMS-like tyrosine kinase 3-ligand (Flt3-L) + TPO but the maximum differentiation was seen when CD133+ cells were expanded in stem span medium with SCF + Interleukin 3 (IL-3) + TPO for the first and in TPO for the second week. Colony Forming Unit-MK (CFU-MK) was formed in three sizes of colonies in the mega-cult medium. In the DNA analysis; 25.2 ± 6.7% of the cells had more than 2n DNA mass. Distinct differences in the MK progenitor cell count were observed when the cells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in the second week. Such strategy could be applied for optimization of CD133+ cells expansion followed by MK differentiation.