Primitive Hematopoietic Stem / Progenitor Cell Proliferation Detected by HALO (an ATP-Based Assay System) Coupled with Flow Cytometric Phenotypic Analysis.

The ability to accurately quantitate primitive hematopoietic stem/progenitor cells in multiple samples of cord blood, bone marrow, or peripheral blood has been hampered by time consuming, laborious assays. Previously we described a culture system consisting of low volume methylcellulose cultures (96 well format) in which different specific growth factor combinations are used to initiate colony formation of distinct progenitor/stem cell subsets present in the abovementioned cell populations. The use of methylcellulose allows the developing colonies to be either 1) dispersed into single cell suspensions for flow cytometric phenotyping of progenitor subsets and/or 2) assayed directly in the well using our HALO (Hematopoietic Assays via Luminescence Output) method to detect the ATP content of even small numbers of viable, proliferating cells. Importantly, the HALO platform is based on progenitor colony formation, and has been previously been applied to basic, clinical and all stages of drug development research. In this system, luciferin/luciferase-mediated luminescence is quantitatively driven by intracellular ATP derived from viable cells. The ATP levels have also been shown to positively correlate with cell proliferation as defined by triated-thymidine incorporation. The ATP released from developing hematopoietic clusters is measured during the exponential proliferation phase and is therefore earlier than the differentiated cells in the classical colony assays. Previously we showed that the number of hematopoietic clusters directly correlates with the relative luminescence units (RLU) in the same cultures. In time-dependent and dose response studies, the number of hematopoietic clusters paralleled RLU, indicating that luminescence can also replace manual counting. Our current studies initially focused on different cell doses of unfractionated human cord blood and bone marrow using growth factor combinations that are relatively specific for the proliferation of human HPP-CFC (high-proliferative potential-colony forming cells) or CFC-BLAST (colony forming cells-blast morphology). A ‘Tpo+SCF+Flt-3 ligand’ growth factor combination stimulated optimal HPP-CFC formation at 21-30 days. However, at just 12 days the HALO and phenotypic characterization appeared optimal to detect these early stem/progenitors. The ‘SCF+IL-6+IL-3’ combination was used to stimulate CFC-Blast formation and was assayed at 7 days. HALO indicated that the degree of proliferation in either growth factor combinations was linear using 2,500, 5,000, 10,000 and 20,000 unfractionated cells initially plated per well in a 96 well plate. However, phenotypic analysis of both HPP and CFC-blast conditions indicated that at higher cell concentrations plated per well (i.e., 10,000-20,000) greater differentiation of primitive cell types occurred. Thus, both CD45⁺ /CD34⁺/ CD133⁺/ CD117⁺ cells and CD45⁺/ CD34⁺/ CD38 ^low cells were markedly reduced in cultures containing >5000 unfractionated cells. Further studies of a similar experimental design except using cell populations enriched for CD 34⁺ or CD 133⁺ are in progress. Our results to this point indicate that HALO used in conjunction with non-differentiative culture conditions (optimal cell concentration per well) can quantitatively, rapidly and non-subjectively assay the presence of primitive stem/progenitor cells in human cord blood and marrow samples.