Megakaryocytic differentiation of human progenitor cells is negatively influenced by direct contact with stroma

Abstract

The recently defined ligand for the Mpl receptor, thrombopoietin (TPO), has been found to be the principal regulatory cytokine of megakaryocytopoiesis. Furthermore, it has been hypothesized that direct interaction between stroma or stroma-derived extracellular matrix (ECM) and human progenitor cells (HPC) may modulate megakaryocytopoiesis. For that purpose, we studied megakaryocytic development of HPC, obtained from patients with hematological malignancies in complete remission or solid tumors without bone marrow involvement, under the influence of megakaryocyte growth and development factor (MGDF, a pegylated, truncated molecule related to the endogenous Mpl ligand). The megakaryocytic development of HPC cultured in contact with a stromal layer (contact cultures) was compared with cultures in which HPC were grown separated from a stromal layer by a microporous membrane (non-contact cultures). A significantly lower number of megakaryocytes (CD41- and CD61-positive cells) was obtained from contact cultures compared to non-contact cultures. Furthermore, the expression of CD42b was higher in non-contact cultures, as compared to contact cultures, indicating an increase in megakaryocyte maturation in non-contact cultures. In contrast, no difference in clonogenic capacity was observed (CFU-GM, BFU-E, CFU-Mk). The possibility that direct contact between HPC and stroma induces the production of a soluble inhibitory cytokine as an explanation for the diminished megakaryocytic development in contact cultures, was excluded by performing transwell experiments in which HPC were cultured in direct contact and in a transwell simultaneously. The percentage of megakaryocytes raised from HPC present in the transwell did not decrease, irrespective of the presence of HPC simultaneous below the transwell in direct contact with stroma. It is concluded that both megakaryocytic development out of HPC and megakaryocytic differentiation is reduced in contact cultures, as compared to non-contact cultures. This is due to modulation by adhesive interactions with stroma, stroma-derived ECM or cytokines bound to the membrane of stromal cells, rather than resulting from the production of diffusible cytokines by stromal cells.