A simple, one-step clonal assay allows the sequential detection of committed (CFU-CM-like) progenitors and several subsets of primitive (HPP-CFC) murine progenitors

Murine bone marrow (BM) cells mere cultured in semisolid medium containing interleukin 3 (IL-3) and high doses of G-CSP. Colonies were counted twice, at day 7 and day 14, and the number of granulocyte/macrophage colony-formin g units (CFU-GM) accurately estimated by the subtraction of day-14 from day -7 colonies, based on the principle that colonies detectable at day 7 and p ersisting beyond day 14 are generated by significantly more immature progen itors. The frequency of colonies relative to their size was determined and used to define subsets of high proliferative potential colony-forming cells (HPP-CFC), Two main groups of HPP-CFC were considered: those generating co lonies of 0.6-1.8 mm of diameter or larger than 1.8 mm, The characterizatio n of these groups showed that they correspond to different functional subse ts of HPP-CFC, The replating ability of colonies was estimated, The percent age of clonogenic progenitors in the S phase of cell cycle was measured by cytosine arabinoside suicide assay. The sensitivity of colonies to 5-fluoro uracil (5-FU) in vitro was determined and their survival after an in vivo t reatment with 5-FU compared with that of colony-forming units in spleen (CF U-S), This technique allowed identification of: A) CFU-GR I; B) relatively mature HPP-CFC, probably corresponding to CFU-S day12; C) more primitive HP P-CFC, relatively resistant to 5-FU in vivo and closely corresponding to CF U-S day 14, and D) very primitive HPP-CFC, resistant to 5-FU in vitro, This simple, rapid, and versatile method allows the detection of a broad range of hematopoietic progenitors in murine BM, from committed progenitors to la rgely quiescent, primitive stem cells, as well as the evaluation of the pro genitors' self-renewal and proliferative potential.