Page 150 - Haematologica March 2020

T. Sun et al.
ered CD34-positive cells after seven or 14 days of cocul- ture were plated in a semisolid medium containing methylcellulose or agar in the presence of a cytokine cock- tail, and their hematopoietic potential was estimated by determining the number of CFU. We thereby observed that CD34-positive cells had fewer CFU in total (CFU- Total), particularly for granulocytes and macrophages (CFU-GM), when cocultured with the ET MSC relative to
those obtained with the HD MSC. There was no signifi- cant difference in the number of CFU for megakaryocytes (CFU-MK) between the two co-cultures (Figure 3A-B). These results indicated that BM-MSC from patients with JAK2V617F-positive ET were deficient in the maintenance of normal hematopoiesis.
Many cytokines influence hematopoiesis. To identify the effectors, we performed ELISA on the supernatants of
ABC
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EF
GH
Figure 2. Bone marrow derived mesenchymal stromal cells (BM-MSC) from patients with JAK2V617F-positive essential thrombocythemia (ET) show enhanced pro- liferation and attenuated apoptosis, senescence, and differentiation. A. Growth curves of BM-MSC isolated from HD (n=12) and patients with JAK2V617F-positive ET (n=12). The ET MSC grew progressively faster than controls. B. Decreased apoptosis of BM-MSC derived from patients with JAK2V617F-positive ET as determined by flow cytometry (control, n=16; ET, n=16). C. The number of β-galactosidase–positive cells were lower in BM-MSC derived from patients with JAK2V617F-positive ET compared to those from control patients (control, n=16; ET, n=16). D. Differentiation potentials of MSC toward adipocytes, osteocytes, and chondrocytes were assessed by Oil Red O, Alizarin Red, and Alcian Blue staining, respectively, after induction for 14–21 days. Representative micrographs of BM-MSC derived from HD, and patients with JAK2V617F-positive ET are shown. Variations in the differentiation between HD (n=12) and ET samples (n=16) were quantified by the staining index described in the Methods section. E. Cell cycle status was determined by flow cytometry. Patients with JAK2V617F-positive ET had less MSC in the G1 phase and more in the S and G2 phases relative to those in the HD controls (control, n=12; ET, n=12). F. NES mRNA expression in BM cells of the controls (n=17) and patients with JAK2V617F-positive ET (n=16). G. NES-positive cells in the bone marrow of an HD control (n =1) and patient with JAK2V617F-positive ET (n=1) shown by immuno- fluorescence. H. BM sections of the controls and patients with JAK2V617F-positive ET immunostained with NES (brown). (control, n=8; ET, n=37; upper panels); BM sections of the controls and patients with JAK2V617F-positive ET immunostained with NES (red) and CD34 (brown). (control, n=8; ET, n=37; lower panels). MSC used in each assay were at passage four (except for those immunostained with NES or NES and CD34). *P<0.05; **P<0.01, ***P<0.001; Data are presented as the mean ± SEM. ET: essential thrombocythemia; HD: healthy donors; n: number of unique donors in each group; IF: immunofluorescence; ns: not significant; SEM: standard error of mean.
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haematologica | 2020; 105(3)