Differentiation of CD34+ cells mediated by thrombopoietin and monocytes /macrophages

D ATRI P; NEGROTTOS; POZNER R.G; PACIENZA N; LANDONI V.I; GOMEZ RM; TORRES O; ISTURIZ M.A; SCHATTNER M

Ginebra suiza

Congreso; XIX Congress of ISTH; 2007

DIFFERENTIATION OF CD34+ CELLS MEDIATED BY THROMBOPOIETIN AND MONOCYTES/MACROPHAGES. L. P. D´Atri1, S. Negrotto1, R. G. Pozner1, N. Pacienza1, V. I. Landoni1, R. M. Gomez2, O. Torres1, M. Isturiz1, M. Schattner1 1Hemostasis and Thrombosis, National Academy of Medicine, Buenos Aires, 2Biological Sciences, National University of La Plata, La Plata, Argentina Introduction: The precise role of monocytes/macrophages (Mo) in the regulation of human megakaryocytopoiesis is poorly understood. We evaluated the effect of conditioned media (CM) from Mo on megakaryocytes (Mk) growth. Introduction: The precise role of monocytes/macrophages (Mo) in the regulation of human megakaryocytopoiesis is poorly understood. We evaluated the effect of conditioned media (CM) from Mo on megakaryocytes (Mk) growth. Introduction: The precise role of monocytes/macrophages (Mo) in the regulation of human megakaryocytopoiesis is poorly understood. We evaluated the effect of conditioned media (CM) from Mo on megakaryocytes (Mk) growth. Methods: Human umbilical cord CD34+ cells were cultured in IMDM supplemented with thrombopoietin (TPO) without (control) or with CM (24hs) from resting (R) Mo or Mo stimulated for 1 hour with LPS, TNF or PMA. After 14 days, cells were counted and frequency of Mk (CD61+) and Mo (CD14+) were determined by flow cytometry. Values are absolute number of cells (media±sem x103, n=10). A one-way ANOVA was used to compare differences (*p<0.01 vs Control; #p<0.01 vs R). Results: Mo increased cell proliferation and this effect was greater when Mo were stimulated by any of the three stimuli evaluated. Because the CD61+ frequency in cultures from resting or TNF-stimulated Mo remained unchanged, the overall number of Mk increased (Control: 58±4; R: 73±6*; TNF: 114±13*#). In contrast, the percentage of CD61+ cells was markedly reduced in cultures from LPS- or PMA-stimulated Mo, thus albeit the increase in cell proliferation, Mk generation was lower (LPS: 40±7*#; PMA: 34±6*#). While in control cultures CD14+ cells was around 5%, CM from Mo enhanced the frequency of monocytic cells being this effect amplified by Mo stimulation (Control:3±1; R:20±3*; LPS:51±9*#; TNF:39±10*#; PMA:35±13*#). Preincubation of LPS-stimulated Mo with 15d-PGJ2, the natural ligand of PPAR-gamma receptor, but not with the synthetic agonist ciglitazone, completely suppressed the LPS-CM mediated inhibition in CD61+ differentiation and the increase in CD14+ cells but not the augment in cell proliferation. Cytokine array studies showed that the increased levels of MCP-1 and GRO chemokines could be involved in LPS effect. Conclusions: Our results show that while resting Mo promotes Mk generation from TPOstimulated CD34+ cells, Mo stimulation triggers a differential CD34+  commitment depending on the agonists involved.