Mutant CALR increases phenotypic HSCs with altered behavior in vitro but no self-renewal advantage in vivo. (A) CALR^del/+ mice show increased frequency of HSCs in the BM. Flow cytometry was performed on BM from mice at 3 to 4 months after poly I:C injection; HSC is defined as Lin^–Sca-1⁺c-Kit⁺CD150⁺CD48^–. (B) CALR^del/+ E-SLAM HSCs have a significantly higher cloning efficiency than WT E-SLAM HSCs. Single E-SLAM cells were defined as successfully forming a clone if assayed to have more than 50 cells at day 7 as assessed by flow cytometry. Both CALR^+/+ and CALR^del/+ represent 72 individual E-SLAM HSCs from 3 independent experiments. P value was calculated from a χ² test. (C) E-SLAM HSCs from CALR^del/+ mice exhibit early cell division in a single-cell in vitro assay. Line graphs show that a significantly higher proportion of E-SLAM HSCs completed first division during the 4 days of single-cell in vitro analysis. Three independent experiments were performed. P value was calculated from a χ² test. (D) E-SLAM HSCs from CALR^del/+ mice form larger clones in vitro. After 7 days in culture, E-SLAM clones (irrespective of size) were analyzed by using flow cytometry, and the average number of cells per clone was measured. (E) BM cells from CALR^del/+ mice show similar repopulating capacity in peripheral blood (PB) of primary competitive transplants. Donor repopulation was assessed by using flow cytometry of nucleated peripheral blood with antibodies for CD45.1 and CD45.2 to distinguish donor origin, Ly6g and Mac1 for myeloid lineages, and B220 and CD3e for lymphoid lineages. Competitive repopulating ability is presented as the percentage of repopulated cells derived from test donor cells among the total number of donor-derived cells (ie, test/[test + competitor (comp)]). Bar graphs that show BM cells from CALR^del/+ mice exhibit comparable repopulating capacity to both myeloid and lymphoid cells in peripheral blood. (F) CALR^del/+ BM primary recipients show similar frequency of donor-derived E-SLAM HSCs compared with the control recipient mice. Flow cytometry was performed using antibodies for CD45.1 and CD45.2 together with antibodies for E-SLAM HSC markers. (G) BM cells from CALR^del/+ mice show similar repopulating capacity in BM of primary competitive transplants. Primary recipients were culled 4 months after transplantation and flow cytometry was performed as above to assess donor competitive repopulating ability in the BM. Bar graphs showing BM cells from CALR^del/+ mice exhibit comparable repopulating capacity to both myeloid (Mye) and lymphoid (Lym) lineages in the BM of the primary recipients. (H) BM cells from CALR^del/+ mice show similar repopulating capacity in secondary competitive transplants. Donor repopulation was assessed by using flow cytometry of peripheral blood as in panel E. Bar graphs showing comparable repopulating capacity to multiple lineages in peripheral blood in the secondary competitive transplantation recipients. Data are shown as mean ± SEM. *P < .05; **P < .01. SSC-H, side scatter.