Acs 1019

HEK293 (gifted and DSMZ, CRL-1573, referred to as 293/293WT) cells were cultured in DMEM/F12 with GlutaMAX (Gibco, 10565018) with 10% filtered FBS (Gibco, 10270106) under 5% CO₂. Cells were cultured at 37°C unless otherwise stated, then 37°C served as a control. In addition to HEK293, we used the following cell types HEK293T (gifted, CRL-3216), HCT-116 (gifted, CCL-247), HeLa (gifted, CCL-2), Jurkat (gifted, TIB-152), K562 (gifted, CCL-243), SK-N-SH (gifted, HTB-11). Cells were sub-cultured when they had reached 70–90% confluence and media was changed every other day. Killing curves were used for all cell lines to determine lowest selection concentration for all selection agents. We used culture recommendations from ATCC when possible. We also used human iPS cells (ATCC, ACS-1019) that were used to generate NPCs by neural induction of iPSCs using dual SMAD inhibition and embryoid body generation (StemCell Technologies; 08581, 05832, 05838)^{77 (link)}, prior to experiments described here the NPCs were cultured in STEMdiff Neural Progenitor Medium (StemCell Technologies, 05833) on Matrigel (Corning, 354234). As well as murine primary NPCs (see section mNPC isolation for further information).

For ATAC-seq experiments, induced pluripotent stem cells (iPSCs) were obtained from ATCC (ACS-1019, DYS0100, male neonate). For CRISPR editing experiments, iPSCs were obtained from Synthego (PGP1-SV1, Male age 55). All validation and QC of cell lines were performed by the supplier. We have not authenticated these cells following receival. Conditions for culturing both cell lines include 5% CO² at 37°C. At the iPSC stage, cells were cultured in StemFlex medium on either Geltrex LDEV-free reduced growth factor basement membrane for the ATCC cell line or iMatrix for the PGP1 cell line. When cells reached 80% confluency, they were passaged using ReLeSR

EV isolation was performed as previously described^{72 (link)}. Briefly, human bone marrow MSCs (BM-MSCs) (ATCC PCS-500-012) and human lung carcinoma cell line A549 (ATCC CCL-185) were cultured in the presence of exosome-depleted FBS (Thermo Fisher Scientific) prior to collection of conditioned medium. Human iPSCs (ATCC ACS-1019), which were cultured in serum-free/xeno-free medium, received full media changes every day and the conditioned medium was collected and stored at 4 °C throughout expansion. Bulk conditioned medium was first spun at 2000×g and then filtered with a 0.22 µm filter to remove cellular debris and larger vesicles. Filtered medium then underwent concentration and buffer exchange via TFF (Spectrum) to isolate EVs. Samples were subsequently aliquoted and frozen at − 20 °C for downstream analysis. The size distribution and concentration of each EV sample was assessed via nanoparticle tracking analysis (NTA) using the NanoSight NS300 (Malvern Panalytical). Prior to analysis the equipment was calibrated per the manufacturer’s protocols. EV samples were diluted in sterile PBS immediately before measurement. The acquired data was analyzed by the instrument’s built-in software.
For isolation of the iPSC EVs for repeat RNA sequencing experiments, EVs were isolated following the methods described above for HIV-1 (U1) EVs.