*2.1. Animals and Primary Mouse HSC (mHSC) Culture*

Animal procedures were conducted using protocol 04504AR approved by the Institutional Animal Care and use Committee of the Research Institute at Nationwide Children's Hospital (Columbus, OH, USA). Mouse HSC (mHSC) isolation was accomplished using standard procedures. Briefly, wild-type male Swiss Webster mice (4–5 wks old; *n* = 5) underwent systemic perfusion with 20–30 mL HBSS under deep anesthesia followed by two steps of perfusion with 15 mL Dulbecco's Modified Eagle's Medium (DMEM) containing 2 mg/mL pronase or 0.5 mg/mL collagenase I, respectively. After this 40 min perfusion technique, livers were removed, diced with scissors and digested in 0.33 mg/mL pronase and 166 U/mL DNase I in DMEM at 37 ◦C for 10 min. mHSC were collected by Optiprep density centrifugation, counted using a hemocytometer, and plated at 106 cells/mL in DMEM/F12

medium containing 20% fetal bovine serum for 1 h. For short-term culture, primary mHSC were switched to serum-free HycloneTM SFM4MegaVir medium (Thermo Fisher Scientific, Waltham, MA, USA) one day after initial seeding and the medium was collected 3 days later (i.e., on Day 4) for EV isolation. For long-term culture, the medium on Day 1 primary mHSC was replaced with fresh DMEM/F12/fetal bovine serum (FBS) and the cells were cultured until Day 10 whereupon they were split 1:5 and the resulting passage 1 (P1) cells were grown in DMEM/F12/FBS cells for 5 days. On Day 15, the medium was removed and replaced with SFM4MegaVir medium which was then collected 2 days later (i.e., on Day 17) for EV isolation.
