Blasticidin selection of hADMPCs transduced with single tet-off lentiviral vector platform.

<p>hADMPCs were transduced with pTRE-EGFP-CMV-tTA-2A-Bsd (CMV) or pTRE-EGFP-EF-tTA-2A-Bsd (EF) at m.o.i. of 250. The cells were treated with 4 µg/mL blasticidin and 1 µg/mL Dox for 2 weeks. Then, the cells were cultured in the absence (Dox (-)) or presence (Dox (+)) of 1 µg/mL Dox for 4 days, and analyzed under a microscope (A) and flow cytometer (B). The cells were treated with 100 nM TSA (TSA), 5 µM 5-aza-dC (aza-dC), or both for 48 h before analyzed by flow cytometer. (C) A representative fluorescence histogram of EGFP. (D) The median fluorescence intensities of the EGFP-expressing populations. Error bars represent the standard error of 3 independent analyses. **, P<0.01 (Student's t test). Scale bar, 200 µm.</p

Differentiation potential of Dox-responsive hADMPCs.

<p>Dox-responsive hADMPCs were differentiated into adipocytes (A, E), osteocytes (B, F), chondrocytes (C, G), and neuronal cells (D, H). (A–D) Phase contrast (ph) and fluorescent (GFP) images. Dox-responsive hADMPCs were differentiated in the absence of Dox (Dox(−)) or in the presence of 1 µg/mL Dox (Dox(+)) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066274#s2" target="_blank">material and methods</a> section. (E–I) Confirmation of differentiated cells by oil red O staining for adipocytes (E), alizarin red staining for osteocytes (F), immunohistochemical staining against collagen II for chondrocytes (G), and immunohistochemical staining against β3-tubulin for neuronal cells (H). The percentages of differentiated cells to each cell type were calculated by the computerized image analysis (I). Cells that were not induced to differentiate (non-induced) were used as a negative control. CMV; hADMPCs transduced with pTRE-EGFP-CMV-tTA-2A-Bsd, EF; hADMPCs transduced with pTRE-EGFP-EF-tTA-2A-Bsd. Scale bar, 50 µm.</p

Schematic drawings of the single lentiviral vectors for tet-off system used in this work.

<p>(A) Gateway-compatible destination vectors containing <i>att</i>R recombination sites flanking a <i>ccd</i>B gene and a chloramphenicol-resistance gene, which allows an easy and rapid shuttling of gene of interest flanked by attL sites into the destination vectors using the Gateway LR recombination reaction. They also have an improved version of tetracycline-controlled transactivator (tTA) linked to the blasticidin resistant (Bsd) gene by the Thosea asigna virus 2A (2A) peptide sequence, whose expression is regulated by the CMV or EF-1α promoter. In the present study, we constructed an entry vector encoding EGFP flanked by attL, resulting in a destination clone, pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd (B). In the absence of doxycycline (Dox), tTA-2A binds to the TRE-Tight promoter and activates EGFP transcription. For more details, see the Results section. CMV pro, CMV promoter; LTR, long terminal repeats; ψ, packaging signal; RRE, rev response elements; cPPT, central polypurine tract; TRE, tet-responsive element; Cm^R, chloramphenicol resistance; tTA, tetracycline-controlled transactivator; Bsd, blasticidin resistance; WPRE, woodchuck hepatitis virus posttranscriptional control element; SIN, self-inactivating. (C) (D) hADMPCs were transduced with pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd at m.o.i. of 250. Four days after transduction, the cells were divided into 2 populations; with 1 µg/mL of Dox (Dox (+)) and without Dox (Dox (-)). (C) Fluorescent and phase contrast images. Scale bar, 200 µm. (D) Log fluorescence histograms of EGFP by flow cytometry analysis. (E) The whole cell lysates from hADMPCs transduced with pTRE-EGFP-CMV-tTA-2A-Bsd or pTRE-EGFP-EF-tTA-2A-Bsd were subjected to western blotting to monitor the cleavage efficiency of tTA-2A-Bsd proteins. A primary antibody against TetR was used to detect either tTA-2A-Bsd (non-cleaved form) or tTA-2A (cleaved form). Asterisk indicates a nonspecific band.</p

Expression pattern of surface cell markers on Dox-responsive hADMPCs.

<p>Dox-responsive hADMPCs after selection by blasticidin were cultured in the absence (Dox(-)) or presence (Dox (+)) of 1 µg/mL Dox for 4 days. Expression of the different surface markers were analyzed by flow cytometry and compared to the expression by a parental hADMPCs. They were stained with PE-coupled antibodies against CD13, CD29, CD34, CD44, CD73, CD90, CD105, and CD166. Histogram of a PE-coupled mouse IgG1 κ isotype control is shown in gray. CMV; hADMPCs transduced with pTRE-EGFP-CMV-tTA-2A-Bsd, EF; hADMPCs transduced with pTRE-EGFP-EF-tTA-2A-Bsd.</p

The efficiency of CMV or EF-1α promoter in hADMPCs.

<p>Lentiviral vectors encoding EGFP under the control of CMV or EF-1α promoter were transduced with hADMPCs at m.o.i. of 25, 50, 100, 250, 500, and 1000, and the cells were analyzed by flow cytometry. (A) The percentage of EGFP-positive hADMPCs transduced with CSII-CMV-EGFP (CMV) or CSII-EF-EGFP (EF). (B) (C) The median fluorescence intensities of the EGFP-expressing populations. (B) hADMPCs transduced with CSII-CMV-EGFP or CSII-EF-EGFP at m.o.i. of 100, 250, 500, and 1000 were analyzed. (C) hADMPCs transduced with CSII-CMV-EGFP or CSII-EF-EGFP at m.o.i. of 1000 were analyzed over a 28 day period. Error bars represent the standard error of 3 independent analyses. **, P<0.01; *, P<0.05 (Student's t test).</p