Loss of PROSER1 leads to exhaustion of HSC activity upon serial transplantations. (A) Bar charts showing fraction of CD150⁺CD48^– long-term HSCs, CD150^–CD48^– MPP, CD150⁺CD48⁺ HPC-2, and CD150^–CD48⁺ HPC-1 cells within the LSK population in 5- to 6-month-old wild-type and PROSER1-deficient animals. Data represent mean ± standard deviation (n = 5-6). ∗∗P < .01 (unpaired 2-tailed t test with Welch correction). (B) Representative FACS plots showing gating strategy⁴⁶ as well as CD150/CD48 surface marker expression in LSK cells harvested from wild-type and PROSER1-deficient animals. (C) Schematic showing serial transplantation assay. Sorted LSK cells from CD45.2⁺ wild-type or PROSER1 KO mice were mixed with equal numbers of sorted CD45.1⁺ LSK competitor cells and transplanted into lethally irradiated primary (1°) CD45.1⁺ recipients. After 20 weeks, 6 × 10⁶ unfractionated nucleated BM cells pooled from primary mice were retransplanted into lethally irradiated secondary (2°) recipient mice. (D) Percentages of CD45.2⁺ cells in periodic peripheral blood samples from 1° recipient mice in B220⁺ B-cell, CD4⁺ T-cell, CD8⁺ T-cell, Mac1⁺Gr1⁺ neutrophilic granulocyte, and Mac1⁺ monocyte/granulocyte cell populations over 20 weeks. Data represent mean ± standard error of the mean (SEM) (n = 8). ∗P < .05; ∗∗P < .01 (unpaired 2-tailed t test with Welch correction). (E) Same as in panel D but for 2° recipient animals. (F,H) Bar charts showing percentages of CD45.2⁺ cells in the indicated terminal differentiated lineages in spleens harvested from 1° (F) or 2° recipient animals (H) after 20 weeks. (G,I) Same as for panels F,H but for hematopoietic stem and progenitor cells in BM harvested from 1° (G) or 2° recipient animals (I) after 20 weeks. Data represent mean ± SEM (n = 8). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 (unpaired 2-tailed t test with Welch correction). HPC, hematopoietic progenitor cell; MPP, multipotent progenitor; ns, not significant; PB, peripheral blood; WT, wild-type.