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. 2009 Jul 14;17(11):1967–1978. doi: 10.1038/mt.2009.151

Figure 3.

Figure 3

The specificity of 2.3ColGFP expression in transduced hMSCs. (a) A map of Lenti2.3ColGFP vector. (b) Comparison of GFP expression in cells transduced with the nonspecific pLL3.7 CMVGFP (top panel) and the osteo-specific Lenti2.3ColGFP (bottom panel) at an early stage of culture (1-week post-transduction using fluorescence microscopy. Original magnification: ×100). (c) Induction of 2.3ColGFP expression with time during in vitro osteogenic differentiation. Top and bottom panels show photomicrographs under bright field and fluorescence, respectively. Original magnification: ×40. (d) FACS analysis of GFP expression in hBMSCs transduced Lenti2.3ColGFP vector at day 24. (e) Association of 2.3ColGFP expression with other typical osteoblast differentiation markers. qRT-PCR was carried out to evaluate the pattern of expression of BSP, osteocalcin, and osteopontin. Total RNA was extracted from 2.3ColGFP-transduced hBMSCs cultured in osteogenic medium at 0, 6, 10, 15, and 18 days of culture. (f) Regulation of the 2.3ColGFP expression by osteoblast stimulation and inhibition factors. Human BMSCs transduced with Lenti2.3ColGFP cells were treated with either BMP-2 at 500 ng/ml or PDGF at 10 ng/ml for 2 days when culture became subconfluent. BMP-2, bone morphogenetic protein-2; BSP, bone sialoprotein; FACS, fluorescence-activated cell-sorting; GFP, green fluorescent protein; PDGF, platelet-derived growth factor; qRT-PCR, quantitative reverse-transcription–PCR.