Isolation and Cryopreservation of T Cell-Depleted HSCs

We prepared the HSCs as previously described^{38 (link)}. Briefly, we collected BM from sex and MHC-matched MCMs (Table 1) by aspirating up to 5 mL from four sites, 20 mL total, and removed red blood cells using ACK lysis buffer (Thermo Fisher Scientific). Next, we washed the cells twice with phosphate-buffered saline and incubated them with anti-TCRα/β allophycocyanin (APC) antibodies (clone R73, BioLegend) for 20 min, followed by incubating with anti-APC microbeads (Miltenyi Biotec) for an additional 20 min at 4 °C. Finally, we passed the stained cells through two LS columns (Miltenyi Biotec) stacked on top of one another, collecting both negative and positive cell fractions, and cryopreserving them at 30 million/mL in serum-free expansion medium (SFEM; Stem Cell Technologies) containing 5% fetal bovine serum and 10% dimethylsulfoxide (DMSO) until further use.