Cell culture
HEK293T cells were purchased from GeneHunter and cultured on collagen-coated dishes (Nunc) in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and streptomycin, and 2.5 ng/ml amphotericin B (Gibco).
Immortalized hAEC line cells were previously established in our laboratory and used as donor cells in this study. Briefly, primary human amniotic epithelial cells (hAEC) were isolated from donated healthy human placentae. These hAECs were immortalized by using an SV40 Lentiviral vector (pLenti-SV40-T+t, Applied Biological Materials Inc., Richmond, BC Canada)28. Immortalized hAECs (iAECs) were cultured in DMEM supplemented with 10% fetal bovine serum (Nichirei), 200 μM L-glutamine (Gibco), 1 × nonessential amino acids (Gibco), 5.5 µM 2-mercaptoethanol (Gibco), 5 ng/ml EGF (PeproTech), 100 U/ml penicillin and streptomycin, and 2.5 ng/ml amphotericin B.
All cells were cultured in 5% CO2 at 37 °C.
Lentiviral vector production
Lentiviral vectors (transfer plasmids) were constructed by cloning each fluorescent gene into a backbone of a common second generation lentiviral vector plasmid with hPGK promoter (pLKO). Two human COXA8 genes were inserted under the promoter as a tandem MTS (Fig. 1).
For lentiviral particle production, HEK293T cells were seeded on collagen-coated 10 cm dishes at 70–80% confluency. Cells were cotransfected with 4 μg of lentiviral vector plasmid along with 4 μg psPAX2 and 2 μg of pMD2. G (packaging plasmids, Addgene plasmids 12260 and 12259). Transfection mixtures were prepared in 500 μL of Opti-MEM I (Life Technologies) containing the DNA and 27 μL of Lipofectamine 3000 (Life Technologies) according to the manufacturer's protocol. The medium was changed 12 h after transfection, and lentiviral supernatants were collected after 24 and 48 hr. Collected lentiviral supernatants were concentrated via ultracentrifugation, and the medium was exchanged with phosphate buffered saline.
Imaging
To image mitochondria-targeted proteins, iAECs were seeded on a 24-well plate (Nunc) at a density of 50,000 cells/well and infected with lentivirus after 12 hours. Cells were seeded on glass bottom dishes (Matsunami) 2 days before imaging. Cells were loaded with 100 nM MitoTracker green or Orange CMTMRos (Life Technologies) and 500 ng/ml Hoechst 33342 for 30 min in culture medium at 37 °C. For lysosome staining, cells were loaded with 100 nM LysoTracker Deep Red (Life Technologies) in addition to the above. Then, the cells were washed twice with medium and imaged in 5% CO2 at 37 °C in the culture medium.
For the mitochondrial transfer experiment, HEK293T cells transduced with cytosolic GFP were seeded on collagen- and poly-L-lysine-coated glass-bottom dishes for two days. The cells were treated with 300 μM hydrogen peroxide for 60 minutes at 37 °C and then cocultured with iAECs transduced with mitochondria-targeted TurboRFP in fresh iAE medium. After 20 hours of culture, the cells were loaded with MitoTracker Deep Red (Life Technologies) and imaged at room temperature.
Fluorescence images were captured using a confocal microscope (TCS SP8, Leica) equipped with a 63× objective (NA 1.40, HC PL APO, Leica) with excitation/emission wavelengths (nm) of 405/415–455 for Hoechst 33342, 488/496–543 for MitoTracker Green and EGFP, 555/565–620 for MitoTracker Red and the red fluorescent proteins, and 638/570–700 for LysoTracker Deep Red and MitoTracker Deep Red.
Cell growth and flow cytometry experiments
iAECs were seeded on a 24-well plate at a density of 5x104 cells/well and infected with lentivirus at an MOI of 5. After 4 days of infection, the cells were passaged and cultured for 6 days to exclude virus toxicity. Then, the cells were reseeded on 96-well plates (Nunc) at a density of 5,000 cells/well. Cell proliferation was measured 12, 24, 48, 72, and 120 hours after seeding by using a Cell Counting Kit-8 (Dojindo) according to the manufacturer's protocol.
For flow cytometry analysis, cells were analyzed by a FACS Aria (BD) equipped with 488 (for EGFP) or 561 (for DsRed, mCherry, TurboRFP and mKOκ) nm laser and 530/30 (for EGFP) or 582/15 (for DsRed, mCherry, TurboRFP and mKOκ) nm emission filter. Cells were analyzed 6, 14, 21, 28 and 42 days after infection. Cells were passaged at intervals of 1 week.