International Journal of Cell Cloning, Vol 1, 377-388, Copyright © 1983 by AlphaMed Press
Clonal growth of human megakaryocytic progenitor cells in a micro-agar culture system: simultaneous proliferation of megakaryocytic, granulocytic, and erythroid progenitor cells (CFU-M, CFU-C, BFU-E) and T-lymphocytic colonies (CFU-TL)
D Geissler, G Konwalinka, C Peschel, J Boyd, R Odavic and H Braunsteiner
A simple and reproducible micro-agar culture technique for cloning human
CFU-M is described. Human bone marrow mononuclear cells were suspended in
agar and incubated for 12 days. Stimulation was provided by the direct
addition of phytohemagglutinin-P (PHA-P), erythropoietin (Epo) and
2-mercaptoethanol (2-ME) to the liquid overlayer. A shift from BFU-E and
CFU-C proliferation to CFU-M and CFU-TL was observed with increasing PHA
concentrations. Under optimal conditions (PHA 50 micrograms, Epo 1.2 IU,
2-ME 2 x 10(-4) M, 1% purified BSA, 0.04% human transferrin, saturated with
Fe C13) a linear relationship between colonies formed and plated cell
number were observed. For the routine morphological analysis, the whole
agar layers were stained using the Pappenheim method. For further
characterization of CFU-M, cytochemical stainings and immunofluorescence
tests with rabbit-antihuman factor VIII-related antigen were performed on
the whole agar layers.
