The dominant gain-of-function mutation in the tyrosine kinase JAK2 (1849 G to T) has been detected in over 80% PV patients and in approximately half of ET or PMF patients. The mutation results in a non-synonymous amino acid substitution at position 617 (valine to phenylalanine) in the JH2 pseudo-kinase auto-inhibitory domain. Current models predict that expression of mutant JAK2V617F in patient hematopoietic stem cells leads to activation of signaling pathways important for proliferation and survival. Furthermore, experimental evidence using murine models, in which JAK2V617F is retrovirally over-expressed, display a phenotype that closely resembles PV, including progression to myelofibrosis. Previous reports have indicated that “in vitro” expansion of PV progenitors favors erythroid precursors with decreased JAK2V617F mutational burden (
Exp. Hematol. (2007) 35(4): 587–95
). Investigators in these studies, however, did not report changes in JAK2V617F frequency in sorted live erythroid progenitors after “in vitro” expansion. Furthermore, CD34+cells from PV patients cultured in the presence of 20% serum indicated an inverse relationship between JAK2V617F frequency and Epo dose (Blood (2006) 108(9):3128–34
). However, these reports failed to correlate Epo dose, and JAK2V617F frequency, with live “in vitro” expanded purified erythroid progenitor cells. Using our published procedure (Exp. Hematol. (2007) 35(4): 587–95
), we expanded erythroid progenitors from healthy volunteers and JAK2V617F positive PV patients (mutational burden ranging from 20–90%) in serum-free media supplemented with SCF, Tpo, and Flt3L (100 ng/mL each - week 1) followed by SCF (50 ng/mL), IGF1 (50 ng/mL), and Epo (0, 0.003, 0.03, 0.3, and 3 Units/mL - week 2). Progression of progenitor expansion, erythroid lineage commitment, live/dead cells, and JAK2 mutation frequency was followed by flow cytometry and real-time PCR. At the end of week 2, cells were harvested, stained with anti-CD71 (transferrin receptor), anti-CD235A (glycophorin A), and the Aqua Live/Dead cell reagent. Stained cells were fixed in 2% paraformaldehyde, analyzed, and sorted on a FACSAria flow cytometer. Live, expanded progenitor cells were sorted into CD235A positive and negative populations, and subsequently used for estimation of JAK2 frequency by quantitative real-time PCR (Exp. Hematol. (2007) 35(1): 32–38
). Our results indicate expansion and erythroid commitment of PV progenitor cells in the absence of Epo correlates with the patients’ intrinsic JAK2V617F burden. Furthermore, irrespective of JAK2V617F frequency in purified peripheral blood granulocytes, an inverse correlation between Epo dose and mutation frequency in “in vitro” expanded purified erythroid progenitors was not observed in all patients examined.
