Ultracentrifugation

Generation of hiPSCs was performed as previously described [34 (link)]. Briefly, pMX-retroviral vectors coding for human Oct4, Sox2, Klf4, and c-Myc (Addgene, Watertown, MA), Nfic, and packaging vectors pCMV-VSVG were co-transfected into GP2-293 cells using calcium-phosphate mammalian transfection kit (Takara Bio USA, Inc., San Jose, CA). 48 and 72 hrs after transfection, the virus-containing supernatants were collected, filtered (0.45-mm filter, MilliporeSigma, Burlington, MA), and concentrated through ultracentrifugation (Beckman Coulter, Brea, CA). For the hiPSC generation, hDPSCs were seeded at a density of 1 × 10⁵ cell per well in a six-well plate. 24 hours later, the cells were infected with the virus and maintained with the respective primary cell culture medium in the presence of 8 mg/ml of polybrene (Sigma-Aldrich, St. Louis, Missouri, MO). Five days post-transduction, the cells were reseeded and further cultured in a modified hESC medium after adding 1 mM nicotinamide. The medium was changed every other day until hESC-like colonies emerged. Reprogramming efficiency was quantified with the number of ALP-positive colonies.

CCMV VLPs were re-assembled from insoluble CP aggregates collected from E. coli lysates. To this end, a disassembly-reassembly process was carried out entirely at 4 °C, as follows: E. coli cells induced and incubated overnight were pelleted and resuspended in 5 ml of disassembly buffer (0.020 M Tris-HCl, 0.9 M NaCl, 0.001 M DTT pH 7.4). Next, cells were lysed by continuous ultra-sonication (Sonics Vibra Cell, CT., USA) during 3 rounds of 1 min at 40 % amplitude (output 6). The lysate was centrifuged at 5.000 rpm for 15 min and the insoluble fraction resuspended in 10 ml of disassembly buffer containing 8 M urea and again sonicated but this time at 20 % amplitude using pulses of 10 seconds until the pellet was entirely dissolved. Thereafter another 15 ml of disassembly buffer was added and mixed with the sonication liquid, the sample was centrifuged at 15,000 rpm for 15 min. The supernatant was collected and dialyzed 6 times against dialysis buffer (0.01 M Tris-HCl pH7.4). The soluble CCMV CP fraction was finally reassembled into VLPs during 2 dialysis steps against reassembly buffer (0.1 M NaAc, 0.9 M NaCl, 0.010 M MgCl₂ pH 4.8) and concentrated by ultracentrifugation (Beckman) on a 30 % sucrose cushion at 40,000 rpm for 3 hours. The pellet was resuspended in 1-2 ml of virus buffer (0.1 M NaAc, 0.001 M EDTA, pH 4.8) and stored at 4 °C until further analysis.

The culture media of IFN-γ-primed iMSCs were replaced with phenol red-free DMEM supplemented with 15% EV-depleted FBS as described in our previous study [21 (link),28 (link)]. After 3 days of incubation, the media was harvested and centrifuged for 10 min at 300× g, and the supernatant was centrifuged for 20 min at 2000× g. The supernatant was centrifuged for an additional 80 min at 10,000× g, and the resulting supernatant was filtered through a 0.2 μm vacuum filter (Merck Millipore, Burlington, MA, USA). Finally, exosomes were isolated by ultracentrifugation at 100,000× g for 80 min, and the pellet was subsequently washed with PBS and subjected to ultracentrifugation (Beckman Coulter, Brea, CA, USA). The exosome pellet was then resuspended in PBS.

Exosomes were labeled with Dil (1 μM, Beyotime). The DiI-exosome suspension was incubated at 37°C for 1 h. Excess dye was removed by ultracentrifugation (Beckman Coulter) at 100,000 g for 1 h at 4°C, and the pellets were washed in PBS for 3 times. PBS was used to dilute the final pellets. The labeled-exosomes were added in the supernatant HSCs for 24 h. The uptake was observed by confocal microscopy.