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A Simple, One-Step Clonal Assay Allows the Sequential Detection of Committed (CFU-GM-like) Progenitors and Several Subsets of Primitive (HPP-CFC) Murine Progenitors

Zoran Ivanovi^a, Benedetta Bartolozzi^b, Pietro Antonio Bernabei^b, Maria Grazia Cipolleschi^a, Pavle Milenkovic^c, Vincent Praloran^d, Persio Dello Sbarba^a

^a Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy;
^b Department of Hematology, A.O. Careggi, Florence, Italy;
^c Institute for Medical Research, Belgrade, Yugoslavia;
^d Laboratory of Experimental Hematology, University of Limoges, Limoges, France

Key Words. HPP-CFC • IL-3 • G-CSF • In vitro • Mouse • Bone marrow

Dr. Persio Dello Sbarba, Dipartimento di Patologia e Oncologia Sperimentali, Università di Firenze, viale G.B. Morgagni 50, I-50134 Florence, Italy.

Murine bone marrow (BM) cells were cultured in semisolid medium containing interleukin 3 (IL-3) and high doses of G-CSF. Colonies were counted twice, at day 7 and day 14, and the number of granulocyte/macrophage colony-forming 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 persisting beyond day 14 are generated by significantly more immature progenitors. 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 colonies of 0.6-1.8 mm of diameter or larger than 1.8 mm. The characterization of these groups showed that they correspond to different functional subsets of HPP-CFC. The replating ability of colonies was estimated. The percentage of clonogenic progenitors in the S phase of cell cycle was measured by cytosine arabinoside suicide assay. The sensitivity of colonies to 5-fluorouracil (5-FU) in vitro was determined and their survival after an in vivo treatment with 5-FU compared with that of colony-forming units in spleen (CFU-S). This technique allowed identification of: A) CFU-GM; B) relatively mature HPP-CFC, probably corresponding to CFU-S day12; C) more primitive HPP-CFC, relatively resistant to 5-FU in vivo and closely corresponding to CFU-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 largely quiescent, primitive stem cells, as well as the evaluation of the progenitors' self-renewal and proliferative potential. Stem Cells 1999;17:219-225

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