Genetically Engineered Mesenchymal Stem Cells Stably Expressing
ABSTRACT Objective(s)Mesenchymal stem cells (MSCs) are nonhematopoietic stromal cells that are capable of differentiating into and contribute to the regeneration of mesenchymal tissues. Human mesenchymal stem cells (hMSCs) are ideal targets in cell transplantation and tissue engineering. Enhanced green fluorescent protein (EGFP) has been an important reporter gene for gene therapy. The aim of this study was establishment of MSCs expressing GFP. Materials and MethodsMSCs were isolated and characterized by Immunophenotyping. The pEGFP-N1 plasmid was extracted from previously transformed Escherichia. coli cells and transfected into MSCs using FuGENE HD transfection reagent. Stable cells were established in the presence of geneticin. Expression of GFP was detected by RT-PCR, western blot analysis and immunoflorecent microscope. ResultsMSCs were successfully isolated and characterized. The MSCs transfected with the pEGFP-N1 plasmid expressed GFP both in mRNA and protein levels while cells transfected with empty vector did not. ConclusionThe results suggested that this engineered cell line will be used in the future studies and can easily be traced in vivo.
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ABSTRACT: Objective(s): Ex vivo expansion of hematopoitic stem cells is an alternative way to increase umbilical cord blood (UCB)-CD34+ cells for bone marrow transplantation. For this purpose demineralized bone matrix (DBM) and mineralized bone allograft (MBA) as two scaffolds based on bone matrix and stem cell niche, were simultaneously used to enhance the effect of human mesenchymal progenitor cells (MPCs) - unrestricted somatic stem cells (USSCs) - as a feeder layer. Materials and Methods: USSCs were isolated and characterized by morphological and immunological analysis then seeded on both scaffolds as a feeder layer. UCB-CD34(+) were isolated by MACS method and were co-culture expanded by USSC in 3D and 2D environments. After 3 weeks expansion, cells were counted and were assessed by karyotype, flow cytometry, clonogenic activity, and long-term culture-initiating cells (LTC-IC). Results: Co-culture expansion in DBM and MBA was 29.22-fold and 27.77-fold, no significant differences in colony and LTC-IC were obtained. Maximum number of colonies belonged to the day 14 with the 73% CFU-GM (Colony Forming Unit- Granulocyte/Macrophage) in contrast to the day 0 which was BFU-E/CFU-E (Burst/Colony Forming Unit-Erythroid). Flow cytometry indicated that the percentage of CD34+ marker was decreased in USSC co-culture and the highest percentage was observed in simple 2D culture. Conclusion: Because of acid extraction in the DBM production process, mineral materials were removed and the protein background that was more flexible was presented. Therefore these results suggest that USSC-DBM can be a suitable ex vivo mimicry niche by intensifying of surface/volume ratio and supporting the stem cell differentiation and expansion.Iranian Journal of Basic Medical Science 10/2013; 16(10):1075-87. · 0.24 Impact Factor
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ABSTRACT: The capacity of mesenchymal stem cells (MSCs) to survive and engraft in the target tissue may lead to promising therapeutic effects. However, the fact that the majority of MSCs die during the first few days following transplantation complicates cell therapy. Hence, it is necessary to strengthen the stem cells to withstand the rigors of the microenvironment to improve the efficacy of cell therapy. In this study, we manipulated MSCs to express a cytoprotective factor, heme oxygenase-1 (HO-1), to address this issue. Full-length cDNA of human HO-1 was isolated and cloned into TOPO vector by TOPO cloning reaction. Then, the construct was ligated to gateway adapted adenovirus expression vector by LR recombination reaction. Afterwards, the recombinant virus expressing HO-1 was produced in appropriate mammalian cell line and used to infect MSCs. The HO-1 engineered MSCs were exposed to hypoxic and oxidative stress conditions fol-lowed by evaluation of the cells' viability and apoptosis. Transient expression of HO-1 was detected within MSCs. It was observed that HO-1 expression could protect MSCs against cell death and the apoptosis triggered by hypoxic and oxidative stress conditions. The MSCs-HO-1 retained their ability to differentiate into adipogenic, chondrogenic, or osteogenic lineages. These findings could be applied as a strategy for prevention of graft cell death in MSCs-based cell therapy and is a good demonstration of how an understanding of cellular stress responses can be used for practical applications.Cell Stress and Chaperones 04/2012; · 2.48 Impact Factor
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ABSTRACT: Human umbilical cord blood (HUCB) is now considered as a valuable source for stem cell-based therapies. Previous studies showed that intravascular injection of the HUCB significantly improves neurological functional recovery in a rat model of intracerebral hemorrhage (ICH). In the present study, we hypothesize transplanted HUCB derived mononuclear cells (UC-MCs) can decrease injured volume and also ameliorate neurological function in ICH rats. Experimental ICH was induced by intrastriatal administration of collagenase in rats. One day after surgery, the rats were divided into 3 groups to receive intravenously either BrdU positive human UC-MCs [(4×10(6) and 8×10(6) cells in 1 ml saline, n=10 respectively) as treated groups] or the same amount of saline [as lesion group (n=10)]. There was also one group (control) that received only vehicle solution of collagenase. The animals were evaluated for 14 days with behavioral tests. Transplanted UC-MCs were detected by immunohistochemistry. Histological data and scores of functional tests were analyzed using ANOVA. Cellular co-localization of BrdU+ cells in the histological slides was determined by software Image J. Intravenously transplanted UC-MCs migrated selectively to the hematomal area and reduce injured volume. The UC-MCs transplanted groups showed better performance on functional tests after 2 weeks compared with the lesion and control groups (P< 0.05). There was no difference in the functional recovery and injured volume improvement between the 2 treated groups. Intravenously transplanted UC-MCs accelerate neurological function recovery of ICH rat and diminish the striatum lesion size. Thus these cells may provide a potential cell candidate for cell-based therapy in ICH.Iranian Journal of Basic Medical Science 01/2012; 15(3):860-872. · 0.24 Impact Factor