Old HSC states persist after long-term residence in young BM niches. (A–E) Age-associated phenotypes of old HSCs residing in separated Y Het mice (sO/Y HSCs) with (A) experimental setup for postparabiosis separation (3/24 cohorts); (B) frequency of crossover over time in blood (left), BM (middle), and HSC (right) compartment at the time of separation (Y-Het) and 4.5 mo afterward (sY-Het); (C) O/Y HSC BM frequency before and after separation; and (D) CD150 levels and (E) regenerative capacity following transplantation into lethally irradiated recipients (250 HSCs/recipient) for the indicated HSC populations. Both sY-Het HSCs and sO/Y-Het HSCs were isolated from individual sY-Het donor mice and transplanted into two to three recipients each. Y HSCs were isolated from two pooled young donor mice and transplanted into three recipients. (F–I) Heterochronic transplantation of 2,000 young or old BJ HSCs into sublethally irradiated young or old B6 recipients (recip.) with (F) experimental setup; (G) blood engraftment over time; (H) BM HSC chimerism at 4 mo after transplantation; and (I) regenerative capacity following transplantation into lethally irradiated secondary recipients (500 HSCs/recipient) for the indicated HSC populations. Data are means ± SD; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.