Megakaryocyte Growth and Maturation from Purified Peripheral Blood Progenitors in Unilineage Serum-Free Liquid Culture

Abstract

Hematopoietic progenitor cells (HPCs), 80–90% purified from peripheral blood, were induced to megakaryocyte differentiation/ maturation in serum-free liquid suspension culture treated with a hematopoietic growth factor (HGF) cocktail (IL-3, KL and IL-6) and/or recombinant mpl ligand (thrombopoietin, Tpo). Particularly, (i) the HGF cocktail induced the growth of a 40% megakaryocyte (MK) cell population, i.e., 4x104 cells at day 0 generated 2x105 MK at terminal maturation; (ii) further addition of Tpo increased the MK purity level to 80% with a final yield of 4x105 MKs; (iii) treatment with Tpo alone resulted in a 97–99% MK population with a mild increase of cell number (up to 1x105 cells). In all culture conditions, morphological evaluation indicated the presence of putative mononuclear MK precursors and then mature polynucleated MK peaking at day 5 and 12 respectively. Membrane phenotype analysis showed a gradual decline of CD34+ HPCs, coupled with an inverse rise of MK-specific antigens (e.g., CD61/62/42b) starting prior to MK detection by morphology analysis. In situ hybridization showed the expression of MK-specific von Willebrand gene in both immature and mature MKs.