Clonal origin of murine hemopoietic colonies with apparent restriction to granuclocyte-macrophage-megakaryocyte (GMM) differentiation

Abstract

We characterized murine hemopoietic colonies consisting of granulocytes, macrophages, megakaryocytes, and blast cells and yet lacking erythroid elements. Mouse marrow or spleen cells were cultured in methylcellulose media in the presence of 10% (v/v) pokeweek mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) and 2 units/ml erythropoietin for 8 days. Granulocyte-macrophage-megakaryocyte (GMM) colonies could be distinguished from granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies because the former lacked the typical appearance of bursts with red color. Analysis of Y-chromosomes in mixing experiments with male and female marrow cells confirmed the clonal nature of the GMM colonies. Differential counts of GMM colonies revealed varying, but significant, numbers of blast cells in all of the day-8 and day-12 colonies and in seven out of ten day-14 GMM colonies. In general, the percentages of blast cells were inversely related to the length of incubation in culture. Replating experiments confirmed the absence of late erythroid precursors such as CFU-E and normoblasts in all of the 50 day-8 GMM colonies. However, six out of the 50 GMM colonies contained early progenitors capable of erythroid expression, such as BFU-E, CFU-EM, CFU-GEM, and CFU-GEMM. In contrast, the three day-14 GMM colonies which did not reveal blast cells failed to produce secondary colonies. Thus, while the progenitors for the latter colonies are restricted to only granulocyte-macrophage-megakaryocyte differentiation, some of the apparent GMM colonies containing blast cells may have originated in early progenitors close to pluripotent stem cells. Detailed cytological analyses and replating experiments are necessary for characterization of true differentiation potentials of mixed colonies in culture.