Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors

Abstract

We analyzed the differentiation of murine hemopoietic colonies derived from paired progenitors in culture. Single progenitors were isolated by use of a micromanipulation technique from blast cell colonies cultured from the spleens of 5-fluorouracil-treated mice. Eighteen to 24 hr later, the paired progenitors were separated with a micromanipulator and cultured in methylcellulose medium containing erythropoietin and pokeweed-mitogen spleen cell conditioned medium. Six to nine days later, the two colonies derived from the paired progenitors were individually picked and differential counts were performed by using May-Grunwald-Giemsa stain. The abbreviations used here are n, neutrophil; m, macrophage; e, eosinophil; mast, mast cell; M, megakaryocyte; E, erythrocyte. Of a total of 387 pairs that could be evaluated, 68 were pairs of colonies consisting of dissimilar combinations of cell lineages such as m-nmmastEM, M-nmmastEM, nm-nmmastEM, nmmastM-nmmastEM, M-nmmastM, nmmast-nmmastM, nm-nmmastE, M-nmM, n-nmM, mM-nmM, m-nmmast, nm-nme, me-nm, mM-nm, n-ne, m-mmast, m-mM, M-nm, M-mM, E-nm, m-nm, M-m, etc. Thirty-nine were homologous pairs revealing identical lineage combinations such as nmmastEM, nmmastM, nmmast, mmastEM, nmEM, nme, nmM, mM, and nm lineages. However, in members of some of these pairs, the proportions of the individual cell lineages were significantly different. The remainder were pairs of single lineage colonies. Paired progenitors obtained from the stem cell colonies of normal mice also revealed homologous and nonhomologous expression of the cell lineages. Comparison of lineage expression in colonies derived from single progenitors with the sum of lineages expressed in pairs of colonies derived from single progenitors indicated that the diversity was not due to injury inflicted by micromanipulation. These observations provide experimental data in support of stochastic mechanisms of stem cell differentiation.