Regulation of thrombopoietin-stimulated megakaryocytopoiesis

SCHATTNER M; LEFEBVRE P; SPANIER MINGOLELLI S; GOOLSBY CL; MOSIMAN V; MEHL P; FOSTER D; COHEN I

Vancouver, Canada

Congreso; 2nd International Conference of the International Society for Hematotheraphy and Graft Engineering (ISHAGE); 1995

Following the cloning of the c-mpl ligand (thrombopoietin or TPO), the requirement of additional cytokines for the TPO optimal stimulation of megakaryocytopoiesis (MKP) has been controversial. We investigated the effect of IL-3 on TPO stimulation of MKP in a liquid culture system using human bone marrow non-adherent mononuclear cells (MNC) and positively selected CD34+ cells. Since we were unable to grow megakaryocytes (MKs) in a serum-free medium containing TPO in the presence or absence of IL-3, IL-11, SCF and IL-6, cultures were carried out in lscove’s medium supplemented with 2.5 % of normal human serum obtained from platelet-free plasma. Tho concentration of TPO which yielded the maximal concentration of MKs was 50 U/ml. The relative frequency of CD34+ and CD41a+ cells as well as ploidy were determined by flow cytometry. Results were expressed in absolute number of MKs (CD41a+ cells) obtained per 1000 seeded CD34+ cells. While in CD34+ cultures lL-3 (100 U/ml) in combination with TPO resulted in a significantly higher number of MKs (7,833 ± 784 SEM) than that obtained In the presence of TPO alone (1,482 ± 524 SEM), no difference was observed in MNC cultures supplemented with TPO alone or TPO with IL-3 (11,739 ± 3,049 vs. 14,000 ± 2,327 SEM, respectively). These results additionally show that MNC yielded higher MK concentrations than CD34+ cells when TPO was used in the absence or presence of IL-3. MKs from cultured MNC exhibited in all conditions higher ploidy patterns than those obtained from cultured CD34+ cells. We also observed that CD34+ cells yielded higher MK numbers when either co-cultured with an irradiated bone marrow stroma or separated from it by a semi-permeable membrane than when cultured in a stroma-free system. These results indicate that factors from accessory and stromal cells are necessary for the TPO stimulation of MKP.