Figure 5.

JAK inhibitors have little effect on self-renewal of CD34+ progenitor cells. Hematopoietic progenitor cells generated from spin-EB differentiation of PVB1.4 induced pluripotent stem cells (iPSCs) were cultured in serum-free medium supplemented with TPO, stem cell factor, and Flt3-L for 7 days. JAK2 inhibitors at indicated concentrations were added throughout the culture. (A): Total cell number in each condition was counted and divided by the input cell number to yield the fold of expansion (n = 2). (B): In the same experiments as shown in (A), cells were also analyzed by flow cytometry and the percentages of CD34⁺ cells were compared among all culture conditions. (C): Cells cultured with DMSO, 250 nM INCB018424, TG101348, or CYT387 were plated in methylcellulose medium for 14 days. Colonies with myeloid and erythroid morphology were numerated. (D): Purified CD34⁺ cells from day 14 of spin-EB differentiation of iPSCs, and the cells further differentiated for additional = days in either erythroid culture condition (EPO) or in progenitor culture condition (TPO) were harvested and analyzed by quantitative polymerase chain reaction for JAK2 expression. JAK2 expression in both cell types was first normalized by β-actin expression then normalized to that of the cells cultured in erythroid condition for 5 days (n = 3). Data are mean ± SD. *, p < .05. Abbreviations: DMSO, dimethyl sulfoxide; EPO, erythropoietin; TPO, thrombopoietin.