Increased in vitro amplification of mutated erythroid CD34⁺CD38⁺ progenitor cells from ET and PV patients. Bone marrow CD34⁺CD38⁺ cells from patients and healthy subjects were cultured in serum-free medium containing SCF, IL-3, dexamethasone, and 1 IU/mL Epo. On day 7, cells were enumerated and phenotyped by MGG and CD36/GPA staining, which demonstrated that more than 90% of the cells belonged to the erythroid lineage (data not shown), and the JAK2 617V>F/total JAK2 ratio was determined by real-time PCR. CD34⁺CD38⁺ cells from patients were seeded in both methylcellulose and liquid culture assays. Methylcellulose assay in the presence of 1 IU/mL Epo was performed to determine JAK2 617V>F/total JAK2 ratio in the input erythroid progenitor cells of the liquid culture at day 0. Erythroid colonies were randomly picked and genotyped on day 14. The JAK2 617V>F/total JAK2 ratio was retrospectively calculated using the formula (h + 2H) /(2h + 2H + 2w) where h, H, and w are the respective numbers of heterozygous, homozygous 617V>F, and wild-type colonies. (A) In vitro amplification of bone marrow CD34⁺CD38⁺ cells from 2 ET patients (▩, ET1 and ET2), 3 PV patients with a JAK2 617V>F homozygous profile (■, PV2, PV5, and PV6), 2 PV patients with a heterozygous profile (▨, PV1 and PV10), and 3 healthy subjects (□, N1, N2, N3) in serum-free medium containing SCF, IL-3, dexamethasone, and 1 IU/mL Epo. Note that, on day 7, cells from patients expanded significantly more than cells from healthy bone marrow (Student t test, P = .001). (B) JAK2 617V>F/total JAK2 ratio in the input erythroid progenitors as indicated (day 0) and in the erythroid progeny after 7 days of liquid culture culture (day 7). ■, PV patients with a JAK2 617V>F homozygous profile; ▨, PV patients with a heterozygous profile; ▩, ET patients.