SINGLE-CELL ORIGIN OF HUMAN MIXED HEMATOPOIETIC COLONIES EXPRESSING VARIOUS COMBINATIONS OF CELL LINEAGES

  • 1 January 1985
    • journal article
    • research article
    • Vol. 65  (4) , 1010-1016
Abstract
Single-cell culture was established for human mixed hemopoietic colonies using a micromanipulator. Mononuclear cells from human umibilical cord blood were cultured at a concentration of 1 .times. 104 cells/ml in methylcellulose medium containing medium conditioned by phytohemagglutinin-stimulated leukocytes and erythropoietin. It was possible to identify the single hemopoietic progenitors in situ in methylcellulose culture on the basis of unique morphology and migratory ability after 36-60 h of incubation. Candidate single hemopoietic progenitors from methylcellulose medium were individually micromanipulated to secondary culture dishes and cultured for an additional 10 to 14 days. The colonies derived from the single progenitors were individually picked and stained with May-Grunwald-Giemsa for analyses of the cellular composition. A total of 288 single cells were individually transferred to second dishes. Then 186 single cells produced secondary colonies consisting of cells in 1-5 different lineages. A total of 39 single cells produced mixed hemopoietic colonies consisting of cells in 2, 3, 4 and 5 different lineages. There were 8 types of colonies revealing 2 different lineages, i.e., neutrophil (n)-erythrocyte (E), macrophage (m)-E-, m-megakaryocyte (M), eosinophil (e)-basophil (b), eE, bE, bM, and EM lineages, were also seen, i.e., nmM, nbE, and ebE. There were 6 types of colonies consisting of cells in 4 lineages, i.e., nmbM, nmEM, nebE, mebE, mebM, and meEM. One type of colony consisted of cells in 5 different lineages (nmbEM). These results indicate the single-cell origin of human mixed hemopoietic colonies expressing various combinations of cell lineages. It also provides experimental data in support of stochastic mechanisms of stem cell differentiation.