Figure 4

The JAK2V617F mutation alters the MK–vascular niche interactions to promote HSPC expansion. (a) There were markedly increased numbers of MKs in Tie2/FF1 recipient mice (right) compared with WT recipients (left). (b) Clusters of MKs were preferentially located near sinusoids (S). (c) Reticulin staining of marrow demonstrates fibrosis in 3/3 Tie2/FF1 recipients, but not in the three examined WT recipients. (d) CD45.2⁺ (JAK2V617F-mutant) MKs (black bar) increased significantly in the Tie2/FF1 mice (n=5) compared to controls (n=5), while there was no significant difference of the CD45.1⁺ (wild-type) MKs (gray bar) between the two groups. (e) The fluorescence image of ECs. DAPI (blue), Von Willebrand Factor (VWF) (green), CD31 (red). (f) JAK2V617F MKs gained a greater growth advantage compared to JAK2WT MKs when co-cultured on a feeder layer of JAK2V617F spleen ECs than when cultured in SFEM alone. JAK2V617F MK cell proliferation was shown as the relative ratio compared to JAK2WT MKs cultured under the same conditions. Data are from two independent experiments with triplicates in each experiment. (g) JAK2V617F MKs promoted CD150⁺CD48⁻ HSPC proliferation more than JAK2WT MKs did in an in vitro direct co-culture experiment. Cell proliferation was shown as fold of expansion which is the ratio of the final cell count to starting cell count. Data are from three independent experiments with triplicates in each experiment. (h) A scratch assay was performed in JAK2V617F spleen ECs. JAK2V617F MKCM significantly stimulated JAK2V617F EC migration compared to that of WT MKCM. The distances from one side of the scratch wound to the other side were measured using ImageJ software (National Institute of Health, Bethesda, MD, USA) at six different locations for each culture condition. The distance of wound closure at time 24 and 48 h was compared with the distance at time 0 h which was set as 1. The results were expressed as the mean±s.e.m. (n=6). Data are from one of two independent experiments that gave similar results.