Separation of murine megakaryocytes and their progenitors on continuous gradients of percoll

Abstract

Murine bone marrow was separated on continuous Percoll density gradients to analyze the distribution of cells of the megakaryocyte lineage. Eighty-seven percent of the recovered megakaryocytes were found in fractions of density less than 1.058 g/cm³, with 63% of these cells found between 1.020 and 1.036 g/cm³. When megakaryocytes were classified according to size, 92% of the large ( ≥ 18 μm) acetylcholinesterase (AchE) positive cells were found in the least dense fractions (1.016–1.039 g/cm³), whereas 86% of the small (⩽ 10.6 μm) AchE positive cells were found in fractions of higher density (1.039--1.078 g/cm³). The distribution of enzymatic AchE activity of the separated fractions corresponded to the location of the histochemically positive cells. When ploidy measurements were made of various fractions, most of the high ploidy (32N and 64N) cells were found at low density (1.028–1.036 g/cm³), whereas no cells greater than 4N were found at density > 1.071 g/cm³. Thus, large AchE positive cells and the cells of highest ploidy were found at lower densities of Percoll, while small AchE positive cells and cells of low ploidy were found at higher densities. An exception to this inverse relationship was found in fractions of lowest density ( > 1.030 g/cm³) where an anomalous distribution of size and ploidy was found. The majority of megakaryocytic colony-forming cells (CFU-MK) were found at high density, as were the granulocyte-macro-phage colony-forming cells (CFU-GM; ∼ 1.074 g/cm³). The density distribution of the incorporation of tritiated thymidine into liquid marrow cultures was concordant with the high density distribution of colony-forming cells. The data show that megakaryocytic maturity and Percoll density varies inversely and that fractionation of marrow on continuous Percoll gradients may be a useful method for the separation and/or enrichment of megakaryocytes at different stages of differentiation.