Gene delivery to human bone marrow mesenchymal stem cells by microporation (original) (raw)
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BMC Biotechnology, 2010
Background: Mesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells.
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.
Genetic engineering of stem cells by non-viral vectors
1st Portuguese Biomedical Engineering Meeting, 2011
Stem/progenitor cells hold a great promise for application in several therapies due to their unique biological characteristics. With the purpose of harnessing these cells full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing efficient and safe methodologies to genetically engineer stem cells, boosting their therapeutic efficacy in Regenerative Medicine. In our work, delivery of plasmid DNA to human Bone Marrow Mesenchymal Stem Cells (BM-MSC) was optimized by lipofection and by a recently available microporation technique and no effect was observed in their immunophenotypic characteristics or differentiative potential. After lipofection similar number of plasmid copies was determined at different cell passages. Importantly, cell proliferation kinetics slowed down due to the presence of plasmid. Overall, we believe our findings are extremely useful towards the maximization of gene delivery to human MSC, without compromising cell function and viability.
Influence of Cell Morphology on Mesenchymal Stem Cell Transfection
ACS applied materials & interfaces, 2018
Gene transfection has broad applications in bioengineering and biomedical fields. Although many gene carrier materials and transfection methods have been developed, it remains unclear how cell morphology including cell spreading and elongation affect gene transfection. In this study, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured on micropatterns and transfected with cationic pAcGFP1-N1 plasmid complexes. Relationship between cell morphology of hMSCs and gene transfection was investigated by using micropatterning technique. Spreading and elongation of hMSCs were precisely controlled by micropatterned surfaces. The results showed that well spread and elongated hMSCs had high transfection efficiency. Analysis of exogenous genes uptake and DNA synthesis activity indicated that the well spread and elongated cell morphology promoted gene transfection through enhanced uptake of the cationic complexes and accelerated DNA synthesis. The results should provide useful ...
Stem Cells, 2006
Viral-based techniques are the most efficient systems to deliver DNA into stem cells because they show high gene transduction and transgene expression in many cellular models. However, the use of viral vectors has several disadvantages mainly involving safety risks. Conversely, nonviral methods are rather inefficient for most primary cells. The Nucleofector technology, a new nonviral electroporation-based gene transfer technique, has proved to be an efficient tool for transfecting hard-to-transfect cell lines and primary cells. However, little is known about the capacity of this technique to transfect adult stem cells. In this study, we applied the Nucleofector technology to engineer human bone marrow-derived mesenchymal stem cells (hMSCs). Using a green fluorescent protein reporter vector, we demonstrated a high transgene expression level using U-23 and C-17 pulsing programs: 73.7% ؎ 2.9% and 42.5% ؎ 3.4%, respectively. Cell recoveries and viabilities were 38.7% ؎ 2.9%, 44.5% ؎ 3.9% and 91.4% ؎ 1.3%, 94.31% ؎ 0.9% for U-23 and C-17, respectively. Overall, the transfection efficiencies were 27.4% ؎ 2.9% (U-23) and 16.6% ؎ 1.4% (C-17) compared with 3.6% ؎ 2.4% and 5.4% ؎ 3.4% of other nonviral transfection systems, such as FUGENE6 and DOTAP, respectively (p < .005 for all comparisons). Nucleofection did not affect the immunophenotype of hM-SCs, their normal differentiation potential, or ability to inhibit T-cell alloreactivity. Moreover, the interleukin-12 gene could be successfully transfected into hMSCs, and the immunomodulatory cytokine was produced in great amount for at least 3 weeks without impairment of its biological activity. In conclusion, nucleofection is an efficient nonviral transfection technique for hMSCs, which then may be used as cellular vehicles for the delivery of biological agents. STEM CELLS 2006;24:454 -461
Optimization of a gene electrotransfer method for mesenchymal stem cell transfection
Gene Therapy, 2008
Gene electrotransfer is an efficient and reproducible nonviral gene transfer technique useful for the nonpermanent expression of therapeutic transgenes. The present study established optimal conditions for the electrotransfer of reporter genes into mesenchymal stem cells (MSCs) isolated from rat bone marrow by their selective adherence to tissue-culture plasticware. The electrotransfer of the lacZ reporter gene was optimized by adjusting the pulse electric field intensity, electric pulse type, electropulsation buffer conductivity and electroporation temperature. LacZ electrotransfection into MSCs was optimal at 1500 V cm À1 with pre-incubation in Spinner's minimum essential medium buffer at 22 1C. Under these conditions b-galactosidase expression was achieved in 29 ± 3% of adherent cells 48 h post transfection. The kinetics of b-galactosidase activity revealed maintenance of b-galactosidase production for at least 10 days. Moreover, electroporation did not affect the MSC potential for multidifferentiation; electroporated MSCs differentiated into osteoblastic, adipogenic and chondrogenic lineages to the same extent as cells that were not exposed to electric pulses. Thus, this study demonstrates the feasibility of efficient transgene electrotransfer into MSCs while preserving cell viability and multipotency.
Biotechnology Letters, 2010
Microporation is an efficient method for delivering plasmid DNA molecules into cultured cells. Herein, we present the optimization of gene delivery by microporation using a Central Composite Design methodology. It was given relevance not only to the transfection efficiency but also to the cell recovery. Different amounts of DNA (1 and 3 g) mainly affected cell viabilities and cell recoveries, which decrease from 93 % to 76 % and from 47 % to 25 % respectively, when higher DNA quantity is used. With this work we suggest an easy methodology to improve transfection of mammalian cells underlining the feasibility to achieve 60% of gene delivery efficiencies whilst recovering 50 % of cells, with 90 % of viability.
International Journal of Molecular Sciences, 2014
Mesenchymal stem cells (MSCs) hold tremendous potential for therapeutic use in stem cell-based gene therapy. Ex vivo genetic modification of MSCs with beneficial genes of interest is a prerequisite for successful use of stem cell-based therapeutic applications. However, genetic manipulation of MSCs is challenging because they are resistant to commonly used methods to introduce exogenous DNA or RNA. Herein we compared the effectiveness of several techniques (classic calcium phosphate precipitation, cationic polymer, and standard electroporation) with that of microporation technology to introduce the plasmid encoding for angiopoietin-1 (ANGPT-1) and enhanced green fluorescent protein (eGFP) into human adipose-derived MSCs (hAD-MSCs). The microporation technique had a higher transfection efficiency, with up to 50% of the viable hAD-MSCs being transfected, compared to the other transfection techniques, for which less than 1% of cells were positive for eGFP expression following transfection. The capability of cells to proliferate and differentiate into three major lineages (chondrocytes, adipocytes, and osteocytes) was found to be independent of the technique used for transfection. These results show that the microporation technique is superior to the others in terms of its ability to transfect hAD-MSCs without affecting their proliferation and differentiation capabilities. Therefore, this study provides a foundation for the
Nonviral Gene Delivery to Mesenchymal Stem Cells Using Cationic Liposomes for Gene and Cell Therapy
Journal of Biomedicine and Biotechnology, 2010
Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from ∼2% to ∼35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and ∼20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.
Human Gene Therapy Methods, 2013
Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources-bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP + cells 48 hr post-transfection, 58 -7.1% and 54 -3.8%, respectively, versus 33 -4.7%. For all cell sources, high cell recoveries (&50%) and viabilities (> 85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP + cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.