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.
Authors: Rich Ivan N. , Hall Karen M. 
Published: April 2020

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.