title: Optimization of high-efficiency transfection of adult human mesenchymal stem cells in vitro.
abstract: 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-beta3-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.
authors.affiliation: Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, National Institutes of Health, 50 South Drive, Rm 1503, Bethesda, MD 20892-8022 USA.
authors.forename: Hana
authors.initials: H
authors.lastname: Haleem-Smith
authors.forename: Assia
authors.initials: A
authors.lastname: Derfoul
authors.forename: Chukwuka
authors.initials: C
authors.lastname: Okafor
authors.forename: Richard
authors.initials: R
authors.lastname: Tuli
authors.forename: Douglas
authors.initials: D
authors.lastname: Olsen
authors.forename: David J
authors.initials: DJ
authors.lastname: Hall
authors.forename: Rocky S
authors.initials: RS
authors.lastname: Tuan
mesh_terms: D000328:Adult; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology*; D064236:Cartilage Oligomeric Matrix Protein; D002454:Cell Differentiation / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D024043:Collagen Type II / Q000096:biosynthesis / Q000235:genetics; D018274:Electroporation / Q000379:methods; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D064235:Matrilin Proteins; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D039902:Multipotent Stem Cells / Q000166:cytology / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: C499177:COL2A1 protein, human; D064236:Cartilage Oligomeric Matrix Protein; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D064235:Matrilin Proteins; C577553:TSP5 protein, human