A Stem Cell Population Capable of Producing Both Lympho- and Hematopoietic Cells Can Be Used To Detect Increased Sensitivity and Residual Stem Cell Damage after Initial Drug Administration.

Abstract Detection of different in vitro stem cell populations has been possible using a number of different parameters, including membrane antigen expression, growth factor combinations, various culture conditions and the ability to produce colonies of specific cell types using the colony-forming differentiation assay. The colony-forming unit-blast (CFU-blast) which is considered to be at the junction point between the lympho- and hematopoietic systems, and the high proliferative potential colony-forming cell (HPP-CFC), regarded as the most primitive in vitro stem cell that feeds into hematopoiesis, are two such populations. Here we describe a primitive stem cell population, that can enter both lympho- and hematopoietic systems, based solely on its proliferative potential. Using a non-subjective, highly sensitive human bone marrow colony-forming proliferation assay that detects and measures changes in intracellular ATP concentration that are proportional to proliferation, a population designated the high proliferative potential stem and progenitor (HPP-SP) cell has been identified. Detection of the HPP-SP, and other in vitro proliferating cell populations (CFC-GEMM, BFU-E, GM-CFC, Mk-CFC, T-CFC, B-CFC), is performed in a 96-well plate format and release of intracellular ATP after lysing the cells drives a luciferin/luciferase reaction to produce luminescence that is detected in a plate luminometer. A variety of drugs, including Imatinib mesylate, 5-fluorouracil, doxorubicin, daunorubicin, hydroxyurea and busulphan were analyzed over a dose response range from 1pM to 10uM. The HPP-SP population was treated with each drug dose and “primed” out of quiescence using IL-3, IL-6, SCF and FL. After 7 days of incubation, 8 replicate 100μl cultures were used to measure proliferation in the primary (1°) stimulated cultures. The cells in a second set of 16 replicates for each dose, were removed from the primary cultures, pooled and re-plated without drug, into secondary (2°), “fully stimulated” cultures containing EPO, GM-CSF, G-CSF, IL-3, IL-6, SCF, TPO, FL, IL-2, IL-7 and IL-15 and incubated for a further 7 days followed by luminescence measurement. All dose response curves were fitted using a 4-parameter logistic statistic. The difference in drug dose response between the 1° and 2° cultures produced a “residual” dose response to the drug and therefore a measurement of how much of the population remained after initial drug treatment. Comparison of the 50% inhibitory concentrations (IC50 values) between the 1° and 2° dose responses indicated whether the remaining HPP-SP population exhibited the same (busulfan) or increased sensitivity (doxorubicin, daunorubicin) to the drug, thereby allowing a prediction to be made as to whether the cells would be further damaged by repeated rounds of drug administration. The cells from the final set of 8 replicate culture were again removed, pooled and analyzed by flow cytometry for the presence or absence of populations resulting from the 2° expansion conditions. These procedures provide a rapid and highly predictive method of ascertaining how primitive stem cells respond to drugs and therefore has a direct implication in chemotherapy and preparation of patients for stem cell transplantation prior to patient treatment.