Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.
We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone.
Blum
Jeremy S
JS
Department of Bioengineering, Rice University, Houston, TX, USA.
Parrott
M Brandon
MB
Mikos
Antonios G
AG
Barry
Michael A
MA
D000256:Adenoviridae / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000187:drug effects* / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D005994:Glycerophosphates / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism; D016212:Transforming Growth Factor beta
C525718:BMP2 protein, human; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D004338:Drug Combinations; D005994:Glycerophosphates; D016212:Transforming Growth Factor beta; C011669:ascorbate-2-phosphate; D003907:Dexamethasone; D008156:Luciferases; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid