The effect of stem cell proliferation regulators demonstrated with an in vitro assay

Abstract

Spleen colony formation after transplantation of bone marrow cells into irradiated mice has been used as an assay for hematopoietic stem cells (CFU-S), but has serious limitations intrinsic to an in vivo assay. In this report we describe experiments using an in vitro clonogenic assay that is especially suitable for studies of stem cell regulation as defined growth factors and normal untreated bone marrow can be used. We have demonstrated that the colony-forming cells have proliferative properties in common with CFU-S and respond to specific proliferation regulators previously detected using the spleen colony assay.

Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

“…To control for any possible effects of the impaired aggregation of this protein on bioactivity, a non-aggregating heparin binding dimeric mutant variant of MIP-la with wild-type activity levels (henceforth referred to as dimeric wild-type MIP-la) was used as a control in all biochemical and functional assays (Graham et al, 1994) including the proteoglycan binding studies outlined in its activity in both the CFU-A stem cell inhibitory assay (Pragnell et al, 1988;Holyoake et al, 1993) and the monocyte shape change assay (Islam and Wilkinson, 1988). Results from the CFU-A assay, an in vitro correlate of the CFU-S assay using either human or murine haemopoietic cells, are shown in Figure 4a and b.…”

“…The CFU-A assay. The murine CFU-A assay was performed as described previously (Pragnell et al, 1988;Lorimore et al, 1990;Graham et al, 1992). Briefly, normal murine bone-marrow cells (5 X 103/ml) were incubated in 0.3% agar in a-minimal Eagle's medium (a-MEM), 25% DHS on top of an underlayer of 0.6% agar/a-MEM/25% DHS with L929 and AF-1 conditioned media as sources of macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM-CSF) respectively.…”

Transforming growth factor beta: is it a downregulator of stem cell inhibition by macrophage inflammatory protein 1 alpha?

“…Direct Addition of TGF-fl and MIP-loz Can Inhibit CFU-A Colony Formation. The CFU-A assay is believed to be an in vitro correlate of the CFU-S hemopoietic stem cell assay (35,36) and thus simplifies the testing of putative stem cell regulatory factors. We have previously reported that direct addition of MIP-lol to the CFU-A assay results in inhibition of CFU-A colony formation (37).…”

“…Bone marrow cells were obtained from female B6D2F1 mice and prepared as suspensions by flushing femora with medium. For the detection of primitive progenitor cells, CFU-A assays were carried out as described previously (35). Briefly, 2 x 104 cells in 4 ml supplemented c~-modified MEM containing 20% donor horse serum and 0.3% agar were seeded on top of an underlayer of the same medium containing 0.6% agar, 10% L929 cell conditioned medium ([CM] as a source of M-CSF), and 10% AF1-19 T cell CM (as a source of GM-CSF) in 45-mm petri dishes.…”

Haemopoietic stem cells: their heterogeneity and regulation

“…During a search for inhibitors of day 12 CFU-S proliferation (Wright et al 1985;Wright & Lorimore 1987;Pragnell et al 1988), we purified an inhibitory activity from the murine bone marrow macrophage cell line J774.2 and sequencing revealed it to be identical to MIP-1a (Graham et al 1990). This protein is active in inhibiting short-term myeloid repopulating stem cells (Broxmeyer et al 1990;Keller et al 1994;Mayani et al 1995) and appears to have little detectable effect on very primitive members of the stem cell compartment (Quesniaux et al 1993;Soma et al 1996).…”