Infection of MSCs by adenovirus expression system expressing NRF2 as a cytoprotective factor

Poor viability of Mesenchymal Stem Cells [MSCs] following transplantation is one of the major challenges in their therapeutic application. Manipulation of MSCs by the genetic engineering method is one of the strategies used to protect the cells against cytotoxic microenvironment. However, maintaining multi differentiation capacity of MSCs following manipulation is important. We investigated if the manipulation of MSCs with NRF2 affects the multi differentiation capacity. MSCs were isolated from bone marrow. NRF2 was isolated and TOPO cloned into the pENTR vector. The recombinant vector was transferred into pAD/CMV/V5-DEST vector by gateway technology. Recombinant adenovirus was produced in AD293 cells, followed by being infected into MSCs. Expression of NRF2 was verified by RT-PCR. The NRF2 engineered MSCs were exposed to stress conditions followed by the evaluation of the cells viability and apoptosis. Finally, NRF2 expressing MSCs differentiation into osteoblast and adipocyte lineages was studied. NRF2 was successfully expressed in MSCs. NRF2- MSCs differentiation into osteoblast and adipocyte lineages indicating overexpression of NRF2 does not affect the differentiation property of MSCs. Expression of NRF2, a well known cytoprotective factor, by using adenovirus expression system does not intervene in the differentiation capacity of MSCs. NRF2-MSCs might be applicable for stem cell-based cell therapy in future

Osteogenic gene expression in osteoblastic differentiation of human mesenchymal stem cells

During differentiation of mesenchymal stem cells [MSCs] into various cells, the expression of a variety of genes undergoes some changes; in this study we decided to investigate the expression rate of some genes like osteopontin [OPN] and osteocalcin [OCN] during this process in order to find a better and faster way for these cells to be differentiated into osteoblasts. In this experimental study, the mononuclear cells of bone marrow were separated and then cultured in DMEM-LG culture media with 10% FBS. During some definite days, the RNA of differentiating cells was extracted. Then, the effective genes in osteogenesis like OPN and OCN were amplified by speciefic primers. The mesenchymal cells were cultured on 3D calcium phosphate scaffolds, and finally the activity rate of the alkaline phosphatase was examined. This research has demonstrated that in the process of differentiation, the expression of the two genes of OPN and OCN changed orderly with the maximum expression of OPN in the 6th day and the maximum expression of OCN in the 7th and 8th days of differentiation. The osteogenic differentiation of MSCs was not confirmed by the coloration of mineral sediments. The activity rate of alkaline phosphatase revealed the preference of 3D calcium phosphate scaffold to 2D environment in this differentiation. The calcium phosphate scaffold positively affects the differentiation process. The expression of OPN and OCN genes changes during differentiation and can be used as away to a better and faster differentiation of these cells into osteoblast

[Application of expired platelets in the preparation of platelet gel and study of proliferative effects of expired platelet derived growth factor on variety of cell lines]

There is growing evidence indicating that growth factors derived from platelets can be used in wound healing. This study aimed to investigate whether old platelets can be used as the main material for preparation of platelet gel and as substitute for FBS and FCS in cell culture medium. In this exprimental study, platelets were prepared from voluntary blood donors by centrifugation. To prove the hypothesis that the platelet gel and the growth factor derived from expired platelets are able to propagate different cells, platelet derived factors were prepared from both new and expired platelet-rich plasma. The concentration of platelet-derived growth factors was measured by ELISA and cell proliferation was measured by MTT assay. The results showed the high quality of platelet gel obtained from old platelets. Our results also revealed that old platelets released growth factors similar to those released by new platelets. The growth factors derived from old and new platelets had the same proliferation effects on MSC, CHO, and Fibroblast cell lines Old platelets released the same growth factors that new platelets did; this showed that old platelets as valuable constituents of blood are cost effective to be used

[Effect on alpha hemoglobin chain expression in K562 cell line]

MicroRNAs [miRNA] are small noncoding RNA molecules that transcribed by RNA polymerase II. After biogenesis, these molecules act by incorporation into the RNA-induced silencing complex [RISC]. MiRNAs are involved in multiple physiological and pathological processes such as proliferation, differentiation, apoptosis and cancer. Recently several studies reported down regulation of mir-150 during erythropoesis. Since hemoglobin expression is valuable indicator of erythroid differentiation we evaluated the mir-150 downregulation effect on alpha chain expression by Quantitative RT-PCR. K562cells were grown in RPMI1640 in standard condition. K562 cells were transfected by microRNA 150 Inhibitor using transfection kit .Mir-150 downregulation was confirmed by miRNA Real time PCR, followed by Q-RT-PCR to investigate the alpha chain expression changes. By relative QRT-PCR the alpha chain expression was increased 10 folds in comparison to untransfected and scramble cells. Furthermore, the differences were statistically significant [P<0.05] Elevation of alpha chain expression in our study showed that mir-150 downregulation has a crucial role in erythroid differentiation and can introduce as a novel marker in alpha thalassemia. Further researches to find out the detail mechanism and miRNAs genes target could improve our knowledge about miRNAs potential in management of diseases and their applications in gene therapy and regenerative medicine

[Effect of growth factors of platelet gel on proliferation and differentiation of mesenchymal stem cells]

Mesenchymal stem cells [MSCs] are bone marrow populating cells, which posses an extensive proliferation potential. In isolation and expansion protocols for clinical scale production of MSCs, fetal bovine serum [FBS] is used as a supplement with potential risk for infections as well as immunological reactions. Autologous platelet gel is made from a natural component of the patient's own blood. Activated platelets release growth factors are mitogenic for MSCs. In vitro studies have indicated that concentration of growth factor varies according to platelet concentration, methods of preparation and mechanism of platelet growth factors release. The aim of our study was to investigate the effect of platelet growth factors on the proliferation and differentiation of human mesenchymal stem cells. Mononuclear cells of bone marrow were collected in 10% FBS growth medium. The expanded cells were characterized by flow cytometric analysis of specific surface antigens. Analysed markers included CD45, CD34, CD166, CD105, CD90, and CD44. The gel is formed by adding calcium and thrombin to platelet rich plasma [PRP]. Treated PRP was incubated for 30 min, 6, 24, 48 and 72 hours in incubator. Growth factors concentrations in supernatants were determined by ELISA. Human mesenchymal stem cells were cultured in the complete medium that supplemented with 10% FBS or Platelet growth factors for 8 days. The rate of proliferation was evaluated by MTT assay. Expanded cells were seeded on calcium phosphate scaffold. Cells growth and morphology on scaffold were analyzed by SEM. Isolation and expansion of MSCs in the complete medium supplemented with platelet growth factors were successful and morphology of cells was compatible with that of FBS. Cells were highly positive for CD90, CD166, CD44 and CD105 and negative for CD34, CD45. There was no significant difference between expression of markers on cells expanded with platelet growth factors and FBS. We demonstrated that platelet growth factors provide a significantly higher proliferative effect on MSCs than those of FBS. MSCs cultured in the presence of growth factors maintain their osteogenic differentiation properties. Osteogenic differentiation was indicated by deposition of mineralized matrix stained with Alizarin red and increased expression alkaline phosphates. Platelet growth factors can be used in place of FBS to provide a safer and more effective culture condition to expand MSC for clinical purposes. MSCs cultured in the presence of platelet growth factors maintain their osteogenic properties

Isolation, cloning, expression and purification of recombinant RhD antigen from cord blood

Rh [Rhesus] is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blood, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study. Total RNAs were extracted from cord blood [O[+]]. The quality of RNA was determined by electrophoresis. In order to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT-PCR. The isolated RhD gene was cloned to pUCIS vector and transformed to DH5alpha. The confirmed construct was sub cloned into expression vector, pBADgIII/A, and expressed in Top 10 E.coli. The expressed protein was characterized by SDS-PAGE and western blot analysis. Antigenicity of the expressed protein was assessed by ELISA using commercially available human anti-RhD polyclonal antibody with peroxidase conjugated goat anti-human IgG, IgM, IgA as secondary antibody. RhD gene was successfully cloned and expressed. The expected size of recombinant RhD protein was detected in SDS-PAGE, and confirmed by dot and western blot analysis. RhD antibody reacted with recombinant RhD antigen as well as with RhD polypeptide extracted from RBCs membrane. The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein and could be applicable for production anti- D antibody in an animal model

[Isolation and detection of RhD antigen from red blood cell [RBC] membrane by immune- precipitation method]

Rh [Rhesus] is a highly complex blood group system in man which plays an important role in transfusion medicine. The aim of this study was the isolation of RhD protein from the membrane of RBCs. In this experimental study immunoprecipitation method with human anti-RhD polyclonal antibody was utilized for the isolation of RhD antigen from Rh[+] human blood samples Proteins of RBCs were characterized by SDS-PAGE and Western blot analysis. Antigenicity of the RhD protein was assessed by ELISA using commercially available human anti-RhD polyclonal antibody with peroxidase conjugated goat anti-human as a secondary antibody. The results show that RhD protein has successfully been isolated by immunoprecipitation method. The expected size of RhD protein was confirmed by Western blot analysis. RhD antibody reacted with RhD antigen prepared from ghost with polyclonal antibody in ELISA, but no reaction was observed in Western blot analysis with monoclonal antibody: It is necessary to mention that this is the primary report of relative purification of RhD and further studies are recommended. The RhD may be helpful to further investigate the molecular basis of RhD protein and could be applicable for production of anti-D antibody in an animal model