Development and Validation of a Novel, High-Throughput Colony-Forming Assay That Allows Simultaneous Detection and Quantitation of Seven Primitive Sheep Hematopoietic Populations.

The hematopoietic colony-forming assays (CFA) allow in vitro detection of clonal expansion of stem, progenitor, and precursor cells, and as such, represent a fundamental tool in the experimental and clinical hematology laboratory. These assays were pivotal to our current understanding of the biology and physiology of the blood-forming system and the process of hematopoiesis. In traditional CFA, cells are suspended in a semisolid medium and are induced by a cocktail of growth factors, which is dictated by the type of cell to be assayed, to proliferate, divide, and differentiate to form colonies that are then viewed under a microscope and manually scored and counted. We have long used this assay in our ongoing experiments in the sheep model as a means of quantifying hematopoietic stem/progenitor cells, but this assay is time-consuming, tedious, rather subjective, and very difficult to implement when large numbers of sheep samples need to be assayed. In the present studies, the traditional sheep CFA methodology was redesigned into an ATP-based bioluminescence proliferation assay readout by modifying and optimizing a newly described assay known as HALO (Hemotoxicity Assays via Luminescence Output [HemoGenix, Inc, Colorado Springs, CO]) to allow measurement of sheep progenitor cells. HALO is rapid, highly sensitive, nonsubjective, standardized, and its 96-well plate format provides high-throughput capability. In these studies, sheep marrow mononuclear cells were isolated with Nycoprep (1.077A), and a 3 parameter HALO cross-over assay was initially performed in 96-well plates for sheep CFC-GEMM using human growth factors and cytokines in an effort to optimize this new system. The three parameters assessed were:

1. Cell dose response (1000, 2500, 5000, 10000, 25000, and 50000 cells/well);

2. Kinetics of colony formation (4, 5, 6, 7, 8, and 11 days); and fetal bovine serum (FBS) concentration (all cell doses and time points were set up at either 5% or 20% FBS).

We also performed a HALO 7-Population assay to detect and measure seven sheep hematopoietic populations, namely two stem cells (HPP-SP and CFC-GEMM), three hematopoietic progenitor cells (BFU-E, GM-CFC, Mk-CFC), and two lymphopoietic populations (T-CFC and B-CFC) at 10,000 cells/well using 5% FBS and 2 readout times of 7 and 11 days. Our result show that 5% FBS is optimal, that cell doses of 5000 to 25000 cells/well all provide equally informative data, and that, at most cell doses, maximal colony proliferation (colony growth) occurs at day 7, making this the ideal time for colony quantitation. All results obtained by HALO were validated by manual scoring and counting, and these two data sets closely paralleled one another, confirming that luminescence readout can substitute for manual inspection and counting. In conclusion, our results show that, as with human, non-human primate, dog, rat, and mouse, sheep bone marrow cells can be stimulated with an appropriate combination of growth factors that allows detection and quantitation of seven different early hematopoietic cell populations at various stages of differentiation ranging from very primitive HSC (HPP-SP) through to committed T and B cell precursors (T-CFC and B-CFC) simultaneously using a sensitive, high-throughput bioluminescence readout-based assay.