Proliferation Versus Differentiation: The Effect of Growth Factor Stimulation Detected by HALO.

In order for stem and progenitor cells to initiate the proliferation process to grow in vitro and form colonies of differentiated cells, growth factors are required. Some factors are specific for proliferation, some for differentiation. Others exhibit both a proliferative and differentiation function. To date, the classical colony-forming assay (CFA) has been used to ascertain the effectiveness of growth factors either individually or in combination. However, the CFA cannot differentiate between a proliferation or differentiation factor because the assay itself is a differentiation assay. We have now developed a unique in vitro system which measures the proliferative capacity of cells to respond to different growth factors. Based on the colony-forming assay, HALO (Hematopoietic Assays via Luminescence Output) measures cell proliferation as a function of the concentration of intracellular ATP which drives a luciferin/luciferase reaction. The end-point of the reaction is bioluminescence detected in a plate reader luminometer. Since the assay does not require cells to differentiate into colonies, no manual enumeration is needed. Furthermore, HALO is a rapid, non-subjective and standardized proliferation assay performed in 96-well plates with high-throughput capability. At least 14 different cell populations with differing proliferative potential from 5 different species can be detected simultaneously. For the present study, both mouse and human bone marrow target cells were used. Eight replicate microcultures of 100ul each were performed to investigate the dose-dependent effect of single growth factors on cell proliferation. The results are summarized as follows. Proliferation of mouse and human hematopoietic cells can be detected between 1 and 2 days and is optimal between 4 and 7 days respectively. The growth factors, SCF, IL-3, IL-6, Flt3-ligand, EPO, TPO and GM-CSF alone, all induce proliferation at less than 1ng/ml. At growth factor concentrations typically used in the colony-forming differentiation assay, many are inhibitory. The EPO dose response for murine CFU-E proliferation measured at 21h produced a flat response and inhibition at doses above 10mU/ml. The mirror image profile was obtained for CFU-E differentiation at 48 h. These results demonstrate that at the CFU-E level, EPO is a differentiation factor and not a proliferation factor. However, EPO alone does stimulate proliferation at the BFU-E stage of development. In conclusion, it is now possible to distinguish between a proliferation and differentiation factor at different hierarchical levels in the hematopoietic system.