THROMBOPOIETIN-STIMULATED EX-VIVO EXPANSION OF HUMAN BONE-MARROW MEGAKARYOCYTES

The effect of thrombopoietin (TPO) on megakaryocytopoiesis (MKP) has b een mainly studied using clonogenic assays in murine systems, In this study, we evaluated MKP in liquid culture using human bone marrow cell s, While interleukin 3 (IL-3) and stem cell factor (SCF) are potent ac tivators of TPO-stimulated MKP in the murine system, only IL-3 exhibit ed synergistic activity with TPO in cultures of human bone marrow. The IL-3 effect on TPO-stimulated megakaryocyte (MK) proliferation, expre ssed as the absolute number of MKs per seeded CD34(+) cell, was more p ronounced with purified CD34(+) cells (8 +/- 1.6 SE versus 2.8 +/- 0.7 SE in the presence and absence of IL-3, respectively) than with monon uclear cells (MNC) (16 +/- 2.8 SE versus 11 +/- 2.0 SE), This effect o f IL-3 on TPO-stimulated MK proliferation was due to a general prolife ration of all cell types since the relative frequency of MKs (32.1 +/- 3 SE and 55.8 +/- 3 SE in MNC and CD34(+) cells, respectively) was no t affected by IL-3, The effect of TPO alone, TPO + IL-3, TPO + SCF, an d TPO + IL-3 + SCF on MK proliferation was examined in MNC and CD34(+) cultures, Greater numbers of MK per seeded CD34(+) were observed in M NC compared to CD34(+) cultures under all conditions except when TPO w as added with both IL-3 and SCF, The enhancing effect of MNC was also observed on MK ploidy in the presence of TPO and IL-3, While prolifera tion and ploidy increase with TPO concentration in the murine system, they are inversely related in the human system, A significant 2.5-fold enhancement of TPO-induced MK proliferation was observed when purifie d CD34(+) cells were cultured in inserts separated from human bone mar row stroma, indicating that soluble stimulatory factors are released f rom the stroma, These observations will be useful for ex vivo expansio n of MKs to treat post-transplant or chemotherapy-associated thrombocy topenia.