Validation and Development of a Predictive Paradigm for Hemotoxicology Using a Multifunctional Bioluminescence Colony-Forming Proliferation Assay

Abstract

The lympho-hematopoietic colony-forming assay has been redesigned into a rapid, nonsubjective and standardized proliferation assay that can measure the effects of compounds on multiple stem and progenitor cell populations from different species simultaneously using a sensitive, high-throughput bioluminescence readout. Eleven reference compounds from the Registry of Cytotoxicity (RC) and eight other compounds, including anticancer drugs, were studied over an 8- to 9-log dose range for their effects on seven cell populations from both human and mouse bone marrow simultaneously. The cell populations studied included a primitive (HPP-SP) and mature (CFC-GEMM) stem cell, three hematopoietic (BFU-E, GM-CFC, Mk-CFC) and two lymphopoietic (T-CFC, B-CFC) populations. The results reveal a five-point prediction paradigm for lympho-hematotoxicity. Depending on how and which populations are affected, the resulting effects in the periphery can be predicted. Validation against the RC Prediction Model produces a high degree of correlation between the in vitro IC(50) values and known in vivo LD(50) values, thereby allowing preclinical dosing to be predicted. If primary human hematopoietic target tissue is used, inhibitory concentration (IC(50)/IC(75)/IC(90)) values of anticancer and other drugs can be converted into predicted clinical doses which, when compared to published chemotherapeutic dosing regimen, are very similar. When performed during early drug screening, the prediction value of the assay should help reduce time and cost, but above all, provide increase efficacy and safety for the patient.

Cord blood banking for clinical transplantation

“…31 Other tests of hematopoietic potency have been proposed, the expression of aldehyde dehydrogenase by MNCs 32 and the levels of ATP after culture in the presence of selective cytokine cocktails. 33 Information on the clinical relevance of these reasonably putative markers is still preliminary.…”

Potency, Proliferation and Engraftment Potential of Stem Cell Therapeutics: The Relationship between Potency and Clinical Outcome for Hematopoietic Stem Cell Products

“…Assay standardization also allows results to be compared over time, a property that is especially important in establishing reference standards within and between laboratories. This readout technology has been incorporated into a stem cell quality assay [13] and two potency assays, one developed for lympho-hematopoietic cells (HALO-96 PQR) and one for mesenchymal stem/stromal cells (LUMENESC-96 PQR). Both assays use the same concepts and principles described here to measure potency and quality and defining acceptance limits for releasing the product for use [14,15].…”

Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation

“…Prior to measuring bioluminescence of the samples after culture incubation, an ATP standard curve was performed (Rich & Hall, 2005;Reems et al 2008;Hall & Rich, 2009). Serial dilutions from a 10M stock concentration were prepared so that the final dilutions were 5, 1, 0.5, 0.1, 0.05, 0.01 and 0.005M.…”

“…It had been previously demonstrated that when hematopoietic stem cells were stimulated to proliferate in the presence of growth factors and cytokines, the intracellular ATP (iATP) concentration increased proportionately to the cell concentration plated (Rich & Hall, 2005;Rich, 2007). The steepness or slope of the cell dose response was dependent upon the primitiveness and proliferation potential of the cells being examined.…”