High-Efficiency Transfection of Human MSCs 9 MOLECULAR BIOTECHNOLOGY Volume 30, 2005 RESEARCH 9 Molecular Biotechnology © 2005 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2005/30:1/009–020/$30.00 Abstract *Author to whom all correspondence and reprint requests should be addressed: Cartilage Biology and Orthopaedics Branch, National Insti- tute of Arthritis, and Musculoskeletal and Skin Diseases, National Institutes of Health, 50 South Drive, Rm 1503, Bethesda, MD 20892-8022 USA. E-mail: tuanr@mail.nih.gov. Optimization of High-Efficiency Transfection of Adult Human Mesenchymal Stem Cells In Vitro Hana Haleem-Smith, Assia Derfoul, Chukwuka Okafor, Richard Tuli, Douglas Olsen, David J. Hall, and Rocky S. Tuan* 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 tech- nique 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% trans- fection efficiency of the viable population of MSCs, using plasmid construct containing a standard cytome- galovirus (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 transfec- tion 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. Index Entries: Transfection; mesenchymal stem cells; GFP reporter; chondrogenesis. 1. Introduction There is increasing interest in the isolation and characterization of multipotent mesenchymal stem cells (MSCs) derived from adult tissues, such as bone marrow stroma (1–3), as well as muscle (4), adipose (5,6), and trabecular bone (7– 10). These MSCs are distinct from hematopoi- etic stem cells, and can be induced to differentiate into various mesenchymal cell lineages, such as osteoblasts, chondrocyte, adipocyte, and myo- blast (11–15). The marrow-derived MSCs are commonly isolated from posterior iliac crest bone marrow aspirates, whereas trabecular bone-derived MSCs are isolated from outgrowths of collagenase- treated bone chips from arthroplasty surgical specimens (7,9). Cells derived from these sources can be cultured and expanded in vitro and retain their ability to differentiate along these multiple mesenchymal pathways (2,15). The MSCs are thus attractive candidate cells for use in tissue engineer-