Stable transfection of MSCs by electroporation.
Human marrow stromal cells (hMSCs) are an attractive source of adult stem cells for autologous cell and gene therapy. To transfect hMSCs without the use of viruses, we developed improved conditions for stable transfection of the cells by electroporation. hMSCs were isolated by adherence to plastic, and were electroporated at 600 V and 100 micros in a 2-mm gap cuvette with a plasmid containing enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase gene (neo(r)). After electroporation of 10(6) cells with 10 microg of the linearized plasmid DNA, hMSCs with stable DNA integration were selected by culturing with 200 microg/ml G418. The transfected hMSCs were expanded another 300-fold in 14 days to obtain 89 million cells, of which 98% expressed EGFP. Chloroquine increased the number of hMSCs transiently expressing EGFP from 12% to over 50%, but decreased stable integration. Stable integration of plasmid DNA into rat MSCs by electroporation was also successful. The transfected MSCs retained their capacity to differentiate into both adipocytes and osteoblasts. Thus, MSCs were stably transfected with plasmid DNA and retained their differentiation capacity after expansion.
Peister
A
A
Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA 70112, USA.
Mellad
J A
JA
Wang
M
M
Tucker
H A
HA
Prockop
D J
DJ
D017667:Adipocytes / Q000166:cytology; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D004247:DNA / Q000008:administration & dosage*; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D010006:Osteoblasts / Q000166:cytology; D051381:Rats; D011917:Rats, Inbred Lew; D014162:Transfection / Q000379:methods*; D019076:Transgenes
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D002738:Chloroquine; D004247:DNA