Efficient Lentiviral Transduction and Improved Engraftment of Human Bone Marrow Mesenchymal Cells (original) (raw)
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Stable Lentiviral Vector Transfer into Mesenchymal Stem Cells In Vivo
Bulletin of Experimental Biology and Medicine, 2015
Green fl uorescent protein (eGFP) gene was transferred into mouse mesenchymal stem cells in vivo using a lentiviral vector. In 2 months after injection of the lentivirus into the cavity of the femoral bone, up to 30% fi broblast CFU in the bone marrow of infected mice contained the alien gene. The transferred gene was found in more than 50% of adherent layers of longterm bone marrow cultures formed by mesenchymal stem cells from the infected mice bone marrow; 4% fi broblast CFU obtained from these layers were labeled. Ectopic hemopoiesis foci developed after transplantation of the bone marrow from infected mice under the renal capsule of syngeneic recipients contained bone tissue labeled with the alien gene in 57% cases and labeled fi broblast CFU in 11%. The data confi rm the possibility of gene transfer with the lentiviral vectors into the mesenchymal stem cells in vivo.
Russian Journal of Genetics, 2012
The efficiency of human bone marrow (BM) mesenchymal stem cell (MSC) transduction with a bicistronic lentivirus vector was estimated, and the stability of transgene expression in genetically modified MSCs was determined. First passage BM MSCs were capable of efficient transduction with the bicistronic lentivirus vector. The transduction efficiency depended on the multiplicity of infection (MOI), being 64.64 ± 6.5 and 88.6 ± 2.9% at MOI 10 and 20, respectively. The lentivirus transduction efficiency proved indepen dent on the number of passages of a BM MSC culture, and expression of the egfp and dsRed1 transgenes in genetically modified MSCs remained stable for one month of culturing. A comparison showed that the level of egfp and dsRed1 transgene expression was preserved upon hepatogenic differentiation in vitro. The results provide a basis for further development of multigenic modification of human BM MSCs for research and/or therapeutic purposes.
Current Gene Therapy, 2011
Mesenchymal stem cells (MSCs) can be isolated from several tissues in the body, have the ability to selfrenewal, show immune suppressive properties and are multipotent, being able to generate various cell types. At present, due to their intrinsic characteristics, MSCs are considered very promising in the area of tissue engineering and regenerative medicine. In this context, genetic modification can be a powerful tool to control the behavior and fate of these cells and be used in the design of new cellular therapies. Viral systems are very effective in the introduction of exogenous genes inside MSCs. However, the risks associated with their use are leading to an increasing search for non-viral approaches to attain the same purpose, even if MSCs have been shown to be more difficult to transfect in this way. In the past few years, progress was made in the development of chemical and physical methods for non-viral gene delivery. Herein, an overview of the application of those methods specifically to MSCs is given and their use in tissue engineering and regenerative medicine therapeutic strategies highlighted using the example of bone tissue. Key issues and future directions in non-viral gene delivery to MSCs are also critically addressed.
Gene Therapy, 2020
Adeno-associated viral vectors (AAV) are unique in their ability to transduce a variety of both dividing and nondividing cells, with significantly lower risk of random genomic integration and with no known pathogenicity in humans, but their role in ex vivo regional gene therapy for bone repair has not been definitively established. The goal of this study was to test the ability of AAV vectors carrying the cDNA for BMP-2 to transduce human mesenchymal stem cells (MSCs), produce BMP-2, and induce osteogenesis in vitro as compared with lentiviral gene therapy with a two-step transcriptional amplification system lentiviral vector (LV-TSTA). To this end, we created two AAV vectors (serotypes 2 and 6) expressing the target transgene; eGFP or BMP-2. Transduction of human MSCs isolated from bone marrow (BMSCs) or adipose tissue (ASCs) with AAV2-eGFP and AAV6-eGFP led to low transduction efficiency (BMSCs: 3.57% and 8.82%, respectively, ASCs: 6.17 and 20.2%, respectively) and mean fluorescence intensity as seen with FACS analysis 7 days following transduction, even at MOIs as high as 10 6. In contrast, strong eGFP expression was detectable in all of the cell types post transduction with LV-TSTA-eGFP. Transduction with BMP-2 producing vectors led to minimal BMP-2 production in AAV-transduced cells 2 and 7 days following transduction. In addition, transduction of ASCs and BMSCs with AAV2-BMP-2 and AAV6-BMP-2 did not enhance their osteogenic potential as seen with an alizarin red assay. In contrast, the LV-TSTA-BMP-2-transduced cells were characterized by an abundant BMP-2 production and induction of the osteogenic phenotype in vitro (p < 0.001 vs. AAV2 and 6). Our results demonstrate that the AAV2 and AAV6 vectors cannot induce a significant transgene expression in human BMSCs and ASCs, even at MOIs as high as 10 6. The LV-TSTA vector is significantly superior in transducing human MSCs; thus this vector would be preferable when developing an ex vivo regional gene therapy strategy for clinical use in orthopedic surgery applications.
Background and Aims: Mesenchymal stem cells (MSCs) are attractive targets for cell and gene therapy, because they can differentiate into many cell lineages. Hence, finding an efficient and suitable method for transferring of genetic materials to these cells is very essential. In this study, we evaluated the efficiency of two methods of gene transferring, viral and nonviral, in transfection of mouse MSCs, comparatively. Materials and methods: MSCs were isolated from mouse bone marrow and their ability to differentiate into osteocyte and adipocyte lineages and their surface markers were evaluated. Then, the efficiency of two methods of nonviral gene transferring; calcium phosphate, and cationic lipid reagents (Lipofectamine™ LTX with Plus™ Reagent and Turbofect TM); and also lentivirus vector were examined in transfection of mouse MSCs with green fluorescent protein (GFP) plasmid. Results: The isolated MSCs successfully differentiated to osteocytes and adipocytes. They were positive for CD24, CD29 and CD44; and negative for CD11b and CD45 cell surface markers. Nonviral gene transferring methods were completely inefficient in transfection of mouse MSCs; whereas calcium phosphate precipitate was completely toxic to mouse MSCs and cationic lipids only transfected less than three percent of the cells. In contrast, high transfection rate and GFP expression (above 70%) was seen with lentivirus vector. Conclusions: it seems that mouse MSCs are refractory to nonviral gene transferring methods. In contrast, lentivirus-mediated gene transfer methods may be an efficient tool for transfection of mouse MSCs without any interference on their phenotype and differentiation potential.
Optimization of High-Efficiency Transfection of Adult Human Mesenchymal Stem Cells In Vitro
Molecular Biotechnology, 2005
With the advent of recent protocols to isolate multipotent human mesenchymal stem cells (MSCs), there is a need for efficient transfection methodologies for these cells. Most standard transfection methods yield poor transfection efficiencies for MSCs (<1%). Here we have optimized a high-efficiency transfection technique for low passage MSCs derived from adult human bone marrow. This technique is an extension of electroporation, termed amaxa Nucleofection™, where plasmid DNA is transfected directly into the cell nucleus, independent of the growth state of the cell. With this technique, we demonstrate up to 90% transfection efficiency of the viable population of MSCs, using plasmid construct containing a standard cytomegalovirus (CMV) early promoter driving expression of green fluorescent protein (GFP). Although little variation in transfection efficiency was observed between patient samples, a 2-fold difference in transfection efficiency and a 10-fold difference in expression levels per cell were seen using two distinct CMV-GFP expression plasmids. By fluorescence-activated cell sorting, the GFP expressing cells were sorted and subcultured. At 2 wk posttransfection, approx 25% of the population of sorted cells were GFP positive, and by 3 wk, nearly 10% of the cells still retained GFP expression. Transfection of these cells with plasmid containing either the collagen type I (Col1a1) promoter or the cartilage oligomeric matrix protein (COMP) promoter, each driving expression of GFP, produced a somewhat lower transfection efficiency (approx 40%), due in part to the lower activity of transcription from these promoters compared to that of CMV. Transfection with the collagen type II (Col2a1) promoter linked to GFP exhibited low expression, due to the fact that collagen type II is not expressed in these cells. Upon culturing of the Col2a1-GFP transfected cells in a transforming growth factor-β3-containing medium known to induce mesenchymal chondrogensis, a significant enhancement of GFP level was seen, indicating the ability of the transfected cells to differentiate into chondrocytes and express cartilage-specific genes, such as Col2a1. Taken together, these data provide evidence of the applicability of this technique for the efficient transfection of MSCs.
Gene Transfer into Rat Mesenchymal Stem Cells: A Comparative Study of Viral and Nonviral Vectors
Stem Cells and Development, 2006
protocols for the treatment of disease and promotion of repair. The efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression with minimal cell death. The study was carried out on bone-marrow derived rat MSCs, and a range of vectors was tested on the same stem cell preparation. Adenovirus, adeno-associated virus (AAV; serotypes 1, 2, 4, 5, and 6), lentivirus, and nonviral vectors were compared. Lentivirus proved to be most effective with transduction efficiencies of up to 95%, concurrent with low levels of cell toxicity. Adenovirus also proved effective, but a significant increase in cell death was seen with increasing viral titer. Rat MSCs remained refractory to transduction by all AAV serotypes, in contrast to rabbit MSCs tested at the same time. Lipofection of plasmid DNA gave moderate transfection levels but was also accompanied by cell death. Electroporative gene transfer proved ineffective at the parameters tested and resulted in high cell death. High and moderate levels of cell transduction using lentivirus vectors did not affect the ability of the cells to differentiate down the adipogenic pathway.
Optimized Lentiviral Transduction of Mouse Bone Marrow-Derived Mesenchymal Stem Cells
Stem Cells and Development, 2008
Mesenchymal stem cells (MSCs) have attracted much attention as potential platforms for transgene delivery and cell-based therapy for human disease. MSCs have the capability to self-renew and retain multipotency after extensive expansion in vitro, making them attractive targets for ex vivo modification and autologous transplantation. Viral vectors, including lentiviral vectors, provide an efficient means for transgene delivery into human MSCs. In contrast, mouse MSCs have proven more difficult to transduce with lentiviral vectors than their human counterparts, and because many studies use mouse models of human disease, an improved method of transduction would facilitate studies using ex vivo-modified mouse MSCs. We have worked toward improving the production of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors and optimizing transduction conditions for mouse MSCs using lentivirus vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G), the ecotropic murine leukemia virus envelope glycoprotein (MLV-E), and the glycoproteins derived from the Armstrong and WE strains of lymphocytic choriomeningitis virus (LCMV-Arm, LCMV-WE). Mouse MSCs were readily transduced following overnight incubation using a multiplicity of infection of at least 40. Alternatively, mouse MSCs in suspension were readily transduced after a 1-h exposure to lentiviral pseudotypes immediately following trypsin treatment or retrieval from storage in liquid nitrogen. LCMV-WE pseudotypes resulted in efficient transduction of mouse MSCs with less toxicity than VSV-G pseudotypes. In conclusion, our improved production and transduction conditions for lentiviral vectors resulted in efficient transduction of mouse MSCs, and these improvements should facilitate the application of such cells in the context of mouse models of human disease.