Chapter 5 —— 122 —— (Thermo Fisher Scientific; Cat. No.: 35050-061) and 5 ng ml-1 Recombinant Human Fibroblast Growth Factor-basic (FGF-2; Peprotech; Cat. No.: 10018B). The hMSCs were kept at 37℃ in a humidified-air 5% CO2 atmosphere. The harvesting of these human primary cells was done following the Best Practices Code of the Dutch Federation of Biomedical Scientific Societies on anonymous surgery material remnants. Recombinant DNA plasmids The AAV transfer plasmids BI17_pAAV-HRS1.A1, BI19_pAAV-HRLMN.A1, and BI38_pAAV-HRA1.IN17 were assembled by using standard recombinant DNA techniques. The complete nucleotide sequences and respective annotated maps of these constructs are available in the Supplementary information. Recombinant AAV productions Recombinant AAV particles were assembled on the basis of BI17_pAAVHRS1.A1, BI19_pAAV-HRLMN.A1, and BI38_pAAV-HRA1.IN17 as follows. HEK293T cells were seeded in T175-cm2 culture flasks at a density of 2×107 cells per flask (up to 18 flasks per AAV vector stock) and, the next day, they were transfected with each AAV transfer plasmid (Supplementary information) together with the packaging plasmid AT51_pDG6.RSV.DsRed. SV40pA mixed at 1:1 molar ratios (30 μg total DNA per T175-cm2 flask). This packaging plasmid expresses the AAV serotype-2 rep and AAV serotype-6 cap genes together with adenovirus helper functions, i.e., VA RNAs I and II, E4ORF6, and E2A (Grimm et al. 2003). Each T175-cm2 culture flask received 99 μl of a 25-kDa linear polyethylenimine (PEI) solution (Polysciences) at 1 mg ml-1 and DNA mixtures, each diluted in 1 ml of 150 mM NaCl. These transfection mixtures were made by dropwise addition of the PEI to the DNA followed by direct homogenization in a vortex for 10 seconds. After 16-18 minutes at room temperature, the resulting DNA-PEI complexes were added to the HEK293T cells with the transfection medium being replaced 24 hours later by 20 ml of
RkJQdWJsaXNoZXIy MTk4NDMw