Rapamycin rescues the self-renewal defect of CD99 Gt/Gt HSCs. (A) Schematic of primary transplants of purified HSCs from CD99 Gt/Gt mice and WT controls followed by treatment of recipient mice with rapamycin or vehicle. (B) Total donor-derived PB chimerism in primary recipients (n = 6 donors and 6 recipients per experimental group) over the course of 24 weeks. (C) Donor-derived myeloid chimerism in the PB over the course of 24 weeks. (D) Frequency of donor-derived HSCs in the BM of primary recipients after 24 weeks. (E) Absolute number of donor-derived HSCs, GMPs, CMPs, and MEPs in the BM of primary recipients after 24 weeks. (F-I) Ex vivo OP-puro incorporation in donor (CD45.1) vs host (CD45.2) HSCs isolated from the BM of recipients at 24 weeks. (J) Schematic of transplants of HSCs purified from primary recipients into secondary recipients treated with rapamycin or vehicle. (K) Total donor-derived PB chimerism in secondary recipients (n = 6 donors and 6 recipients per experimental group) over 24 weeks. (L) Schematic of primary competitive transplants of unfractionated BM from CD99 Gt/Gt and WT mice followed by treatment of recipient mice with CX-5461 (starting 48 hours after transplant and continuing through 10 weeks). (M) Total donor-derived PB chimerism and (N) donor-derived myeloid chimerism in primary recipients (n = 3 donors and n = 7 recipients per experimental group) treated with CX-5461. Statistical significance was assessed using 2-tailed Student t tests for panels B-C,F-I,K,M-N and Mann-Whitney U tests for panels D-E (∗P < .05; ∗∗P < .01; ∗∗∗P < .005; +P < .0005). P values for selected nonsignificant trends are also shown; data are represented as mean ± standard error in panels B-C,K,M-N and mean ± standard deviation in panels D-E,L. IP, intraperitoneal injection.