Pmd2 g

Lentivirus stocks were produced as previously described with slight modifications. Human embryonic kidney 293FT cells (Invitrogen) were transfected using Lipofectamine 2000 (Thermo Fisher) with the expression of two helper plasmids: psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) Ten micrograms of the transfer vector, 5 µg psPAX2 and 5 µg pMD2.G of DNA were used per 10‐cm plate. Forty‐eight hours after transfection, the supernatants of four plates were pooled, centrifuged at 780 × g for 5 min, filtered through a 0.45‐µm pour size filter, and further centrifuged at 24 000 rpm for 2 h. The resulting pellet was re‐suspended in 100 µL of PBS. Lentivirus titration was performed on DIV5‐6 at titer range 10⁷ IU/mL. In most experiments, cultures were infected overnight, rinsed twice with virus‐free medium the next morning and incubated in normal culturing medium for another 48–72 h prior assay.

For the overexpression of Lhx8 or Suv39h1, the open reading frames of target genes were amplified and the amplicon was inserted into the lentivirus expression vector of pWPI (Plasmid #12254; Addgene). A lentivirus was produced through transfecting HEK293T cells with the lentivirus expression vector along with pMD2.G (Plasmid #12259, Addgene) and psPAX2 (Plasmid #12260, Addgene) (pWPI 6.25 μg, pMD2.G 0.625 μg and psPAX2 3.125 μg). Supernatants containing lentivirus particles were then collected after 48 hours and were stored at −80°C before use. DPSCs were infected with a lentivirus in 8 μg/mL polybrene (Santa Cruz Biotechnology). DPSCs were transfected with the lentivirus or the negative control according to Multiplicity of Infection (MOI) 50:1 for 2 days.
For shRNA knockdown of Lhx8 or Suv39h1 in DPSCs, lentiviral shRNAs were purchased from GenePharma. Five shRNAs were used for lentiviral treatment. DPSCs were transfected with the lentiviral‐mediated shRNA or the negative control according to Multiplicity of Infection (MOI) 50:1 for 2 days. Compared with the non‐silencing scramble virus, the shRNAs with the highest knockdown efficiencies were chosen for follow‐up experiments. The chosen sequences were listed in Supplemental Table S1.

EVs isolated from thirty ml of conditioned medium were collected from cells cultured at 70% confluency in two 100 mm plates after 72 h (seeding density 2.2 × 10⁶ cells/plate). The conditioned media was centrifuged at 300 × g for 10 min to remove intact cells, dead cells, and cell debris. The medium was then concentrated using a centrifugal concentrator with a 100,000 molecular-weight cutoff (Amicon^®Ultra-15 Centrifugal filters), yielding about 0.5 ml concentrate (two spins of 10 ml at 6000 × g for 10 min). This concentrate was resolved by passing through IZON qEV original size exclusion columns (SEC) followed by 15 ml of double filtered (0.2 μm) PBS. Five-hundred-microliter fractions were collected. High particle/low protein fractions (from 7 to 11) were pooled and concentrated using Amicon^®Ultra-0.5 Centrifugal filters to a final volume of 200 µL at 10,000 × g for 30 min. The typical yield of an EV isolation was approximately 7.1 × 10⁷ ± 3.2 × 10⁷ particles/ml. This method was adapted to isolate EVs, LVVs (transgene plasmid, psPAX2 (Addgene #12260) and pMD2.G (#12259), VSV G-VLPs (pMD2.G (Addgene #12259), and GAG-VLPs (psPAX2 (Addgene #12260)) before being exposed to scProteins (see below). LVVs purified from media of 2.5 million HEK293Tcells transfected with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) were isolated with SEC.

Transduction retrovirus particles were collected from HEK293T cells following Lipofectamine 3000 transfection of expression plasmids (pLenti CMV TetR BLAST and pLenti X1 shRNA plasmids) and packaging plasmids pMD2.G (Addgene; number 12259; a gift from Didier Trono) and PSPAX2 (Addgene; number 12260; a gift from Didier Trono) according to the manufacturer’s instructions (Invitrogen; L3000015). The overexpression pCT-CD9-RFP (SBI; CYTO123-PA-1) and pCT-CD63-GFP (SBI; CYTO120-PA-1) plasmids were transfected in HEK293T cells with the packaging plasmids pMD2.G (Addgene; number 12259; a gift from Didier Trono), pMDLgpRRE (Addgene; number 12251; a gift from Didier Trono), and pRSVRev (Addgene; 12253; a gift from Didier Trono) according to the manufacturer’s instructions (Invitrogen; L3000015). Medium was collected and reapplied at 48, 72, and 96 h following transfection, centrifuged for 10 min at 1,000 rcf, filtered through a 0.45-μm filter, and frozen at −80°C until use.