Phorbol diesters stimulate the development of an early murine progenitor cell: The burst-forming unit-megakaryocyte

Abstract

When murine (C57BL/6) bone marrow cells are cultivated with WEHI-3 conditioned media, a source of megakaryocyte-colony-stimulating activity (Mk-CSA), and phorbol myristate acetate (PMA), a previously undetected population of megakaryocyte (Mk) progenitor cells is observed. These new Mk colonies are reminiscent of erythroid bursts, in that they contain large numbers (40-500) of Mk and multiple foci (2-7) of development. These burst-forming units, Mk (BFU-Mk), are defined as having ≥42 cells/colony and, at least, three foci of Mk development (colonies grown in soft agar cultures, all studies done at limiting dilutions; colonies detected by acetylcholinesterase [ACh-E] staining). CFU-Mk and BFU-Mk require two activities for optimal growth: Mk-CSA and PMA. However, the BFU-Mk require a tenfold greater concentration of PMA for optimal development (10-6 vs. 10-7 M). BFU-Mk detection is linear (over a range of 25-100 x 103 cells/ml), with the regression line passing through the origin. Bone marrow frequencies of these two progenitor cells are CFU-Mk, 36.7 ± 2.5, and BFU-Mk, 7.3 ± 0.7 per 105 total nucleated cells (mean ± SEM; n = 28). The BFU-Mk have a restricted velocity sedimentation range (3.3-4.5 mmh-1 vs. 3.3-6.8 mmh-1 for CFU-Mk). Modal buoyant densities are 1.068 ± 0.0002 and 1.070 ± 0.002 for BFU-Mk and CFU-Mk, respectively. Thus, these cells are found amoung the smallest and less dense of the Mk progenitors, and are not clumps or clusters of CFU-Mk. Kinetic analysis indicates that CFU-Mk require 5-7 d for optimal growth, whereas BFU-Mk require 10-12 d. Examination of the proliferative potential (cells per colony) shows 19.3 ± 1.5 cells per colony (n = 246 colonies) for day 10 CFU-Mk, vs. 118 ± 6.0 for day 10 BFU-Mk (n = 163). Analysis of the cellularity/subcolony within each burst indicates 37.0 ± 2.1 (n = 146) Mk/colony and 3.9 ± 0.1 subcolonies/burst (n = 100). Finally, >90% of the BFU-Mk contain only ACh-E positive cells, indicating that these are not mixed colonies. These results indicate that the BFU-Mk, compared with the CFU-Mk, require an increased amount of stimulation in order to differentiate, show delayed in vitro development, and have a higher proliferative potential. These data are consistent with the hypothesis that these cells are early progenitor cells in the Mk lineage that antedate the CFU-Mk.