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The effect of thrombopoietin (TPO) on megakaryocytopoiesis (MKP) has been mainly studied using clonogenic assays in murine systems. In this study, we evaluated MKP in liquid culture using human bone marrow cells. While interleukin 3 (IL‐3) and stem cell factor (SCF) are potent activators of TPO‐stimulated MKP in the murine system, only IL‐3 exhibited synergistic activity with TPO in cultures of human bone marrow. The IL‐3 effect on TPO‐stimulated megakaryocyte (MK) proliferation, expressed as the absolute number of MKs per seeded CD34 +  cell, was more pronounced 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 mononuclear cells (MNC) (16 ± 2.8 SE versus 11 ± 2.0 SE). This effect of IL‐3 on TPO‐stimulated MK proliferation was due to a general proliferation 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 not affected by IL‐3. The effect of TPO alone, TPO + IL‐3, TPO + SCF, and TPO + IL‐3 + SCF on MK proliferation was examined in MNC and CD34 +  cultures. Greater numbers of MK per seeded CD34 +  were observed in MNC compared to CD34 +  cultures under all conditions except when TPO was 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 proliferation 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 purified CD34 +  cells were cultured in inserts separated from human bone marrow stroma, indicating that soluble stimulatory factors are released from the stroma. These observations will be useful for ex vivo expansion of MKs to treat post‐transplant or chemotherapy‐associated thrombocytopenia.

Thrombopoietin‐Stimulated ex Vivo Expansion of Human Bone Marrow Megakaryocytes