JAK2V617F induces increased proliferation and differentiation in single HSC-derived clones. (A) Schematic of single-cell in vitro cultures. Single CD45⁺EPCR⁺CD48⁻CD150⁺ (E-SLAM) cells were sorted into individual wells; cultured for 10 to 14 days in STEMSPAN with 10% FCS, 300 ng/mL SCF, and 20 ng/mL interleukin 11; and assessed for proliferation, cell cycle kinetics, and differentiation. (B-C) Daily cell counts revealed that JAK2V617F homozygous HSCs (red line) display faster cell cycle kinetics, as indicated by a shorter time to first and second division, P < .0001 at 48 hours (3 independent experiments). (D-E) Homozygous JAK2V617F HSCs (red bars) give rise to an increased number of differentiated cells (positive for Gr1⁺/Mac1⁺; Lin⁺; P ≤ .0001) and a reduced number of stem/progenitor cells (Gr1⁻, Mac1⁻, c-Kit⁺, Sca1⁺, LSK; P < .0001). WT, n = 100; JAK HET, n = 189; JAK HOM, n = 154 (3 independent experiments). (F) Compared with WT HSCs, JAK HOM HSCs have increased early-forming CAFCs (P = .0086). WT, n = 61; JAK HOM, n = 62 (2 independent experiments). Asterisks indicate significant differences by Student t test for D+E, and by χ-squared for B, C, and F (*P < .05; **P < .01; ***P < .001). Data are shown as mean ± SEM. (G) Principal component (PC) analysis of all HSCs calculated from the 39 genes analyzed. PC1 and PC2 account JAK2V617F HSCs are indicated by red circles, and WT HSCs are indicated by blue diamonds. A cluster of cells on the right hand side of the graph is enriched for JAK2V617F HOM HSCs and have reduced expression of several important hematopoietic genes (Meis1, Smarcc2, Bmi1, Pbx1, Sfpi1, Runx1, Hoxb4, Myb, Lmo2). Axes are in arbitrary units. WT, n = 465; JAK HOM, n = 277.