Haematopoietic stem cell proliferation regulators investigated using an in vitro assay

The in vivo CFU-S murine haematopoietic transplantation assay has allowed haematopoietic stem cell behaviour and regulation to be investigated; however, the in vivo nature of the CFU-S assay restricts its use. Recent use of combinations of haematopoietic colony-stimulating factors (CSFs) in vitro has cloned a population of colony-forming cells from haematopoietic tissue, characterised by high proliferative potential and proposed to be a component of the haematopoietic stem cell compartment. A high proliferative potential colony-forming cell (HPP-CFC) population was assayed from murine haematopoietic tissue using a combination of WEHi 3B myelomonocytic leukaemic cell line conditioned medium (a crude source of interleukin 3 (IL3)/multi-CSF) and L929 fibroblast cell line conditioned medium (a crude source of M-CSF/CSF-1). The proportion of HPP-CFC in S-phase was determined following incubation with an S-phase specific, cytotoxic agent. In normal bone marrow from CBA/H mice, 9% of HPP-CFC were in S-phase, while in sublethally X-irradiated, regenerating bone marrow, 50% of HPP-CFC were in S-phase, a close correlation with in vivo CFU-S kinetics. The kinetic state of appropriate HPP-CFC populations can be modified in vitro by incubation with stem cell specific regulators (inhibitor and stimulator). Both inhibitor and stimulator were titratable against the appropriate target HPP-CFC population. Results obtained showed a close correlation between the in vivo CFU-S and in vitro HPP-CFC titration data, reinforcing the belief that the HPP-CFC population is a developmentally early haematopoietic precursor, possibly a component of the haematopoietic stem cell compartment.