Pcdh cmv mcs ef1 copgfp pcdh

Human BMP2 and VEGF gene primers were designed and amplified as described previously (11 (link),16 (link)). The oligonucleotides were combined into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP (pCDH; System Biosciences, Mountain View, CA, USA) to build the recombinant plasmids, pCDH-BMP2 and pCDH-VEGF. The recombinant plasmids and packaging plasmids were then co-transfected into 293FT cells (Cyagen Biosciences, Inc.). The media of the 293FT cells containing lentivirus was collected 48 h after transfection, and then were purified by ultracentrifugation at 1,000 × g, 37°C for 10 min, and the supernatant was subsequently filtered through a 0.2-µm syringe filter (EMD Millipore, Billerica, MA, USA). The 4th passage SCAP were infected with the cell supernatants which contained lentiviral constructs (multiplicity of infection =70) to obtain SCAP-BMP2 and SCAP-VEGF. Furthermore, SCAP-BMP2-VEGF were generated by infecting SCAP with cell supernatants which contained lentiviral constructs expressing BMP2 and VEGF, sequentially. Serving as the control, a blank vector transfected with SCAP [SCAP-green fluorescent protein (GFP)] was constructed. The expression of BMP2 and VEGF in the four groups of cells was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis four days subsequent to transfection.

Human DLX3 gene primers (PrimerBank ID:38327640c1, Table I) were designed according to PrimerBank online (The Massachusetts General Hospital) (22 (link)). Subsequently, PCR amplification was performed according to our previous study (23 (link)). The amplified DLX3 primers were treated with EcoRI and BamHI, and then combined into the lentivirus vector pCDH-CMV-MCS-EF1-copGFP (pCDH; System Biosciences, LLC) to construct the recombinant plasmid pCDH-DLX3. The target gene was transduced into 4×10⁵ 293FT cells per well in 6 well plate using a combination of enveloping plasmid, packaging plasmid and recombinant lentiviral plasmid (3rd generation system; Cyagen Biosciences, Inc.) at 37°C. Then, 48 h later, 293FT cells were centrifuged at 10,000 × g at 4°C for 4 h and the supernatant was collected to infect the 3rd passage of iPSC-MSCs (iPSC-MSC-DLX3) at 37°C. The MOI was 50, and concentration of Polybrene was 5 µg/ml. Cells were cultured at 37°C in DMEM containing 10% FBS with 1 µg/ml puromycin for 3 days in order to select puromycin-resistant cells and 0.25 µg/ml puromycin was used for maintenance. Similarly, iPSC-MSCs transfected with blank lentivirus vector (iPSC-MSC-GFP) were generated and used as the control. After 2 days, reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis were performed to evaluate the expression of DLX3 in the two groups.