Differential expansion of the HSPCs by MPN disease phenotype and terminal myeloproliferation in human MPNs. (A) Representative fluorescence-activated cell sorter plots are shown for a normal and an MF patient demonstrating the percentage of the lineage⁻/CD34⁺/CD38⁺ myeloid progenitor fraction and lineage⁻/CD34⁺/CD38⁻ fraction, enriched for HSCs. Gates were set according to isotype controls. A bar chart demonstrating the number of Lin⁻/CD34⁺/CD38⁻ cells and GMPs per 10⁶ mononuclear cells is shown (right panels) and revealed no statistically significant increase in the number of these cells in the 3 different MPN compared with normal controls, although there was a trend for an increased HSC compartment in MF versus normals (P = .17). However, there was a statistically significant increase in the CD34⁺/CD38⁻ compartment in MF compared with ET (P = .01) and PV (P = .05). Similar results were obtained when the GMP compartment of patient MF bone marrow was compared with PV and ET patients (P = .05 and .02, respectively). Error bars represent ± SEM. (B) Colony formation for Lin⁻/CD34⁺/CD38⁻ cells is shown for normal controls and MPN patients. Results are expressed as percentage of normal. No significant difference was observed, although there was a trend toward a higher colony number in PV patients versus normals (P = .09). (C) Analysis of differentiation potential of Lin⁻/CD34⁺/CD38⁻ cells in vitro in methylcellulose containing IL-3, SCF, IL-6, G-CSF, GM-CSF, and EPO (3 U/mL). Colonies obtained in panel B were typed, and the average proportions of each colony type are shown. The only differences of statistical significance were between PV and normal controls for granulocyte-macrophage (P = .02) and granulocyte erythrocyte monocyte megakaryocyte (P = .05). Similar findings were obtained at limiting erythropoietin concentrations (0.05 U/mL) (supplemental Figure 1D). (D) FACS plots are shown for a normal and a PV patient, demonstrating the percentage of the maturing CD71⁺/GPA⁺/CD34⁻ erythroid compartment. A bar chart demonstrating the number of CD71⁺/GPA⁺/CD34⁻ cells per 10⁶ mononuclear cells is shown (right panel) and revealed a statistically significant increase in both PV and MF versus normal controls (P = .02, PV vs normal; and P = .05, MF vs normal). To assess the maturing megakaryocyte compartment, CD41⁺/ PI⁺ (to exclude platelets) cells were enumerated, and this analysis revealed a statistically significant increase in PV versus normal controls (P = .04), with a trend toward an enlarged compartment in ET patients (P = .08).