Quantitation of the Proliferative Potential of Highly Enriched Human Primitive Hematopoietic Progenitor Cells Using a Stroma-Free Limiting Dilution Assay with Automated Scoring

Background Increasing use of phenotypically-enriched stem cell populations for clinical hematopoietic transplants has led to an urgent demand for a reliable, rapid and simple functional assay which would provide an estimation of the reconstituting potential of cells prior to transplantation. Methods We have developed a 2-week quantitative, stroma-free assay to measure the frequency of primitive progenitors within hematopoietic cell samples. This relatively short-term assay provides frequency information which correlates with that measured by a 5-week stroma-dependent CAFC assay. Cells with the phenotype CD34 + Thy-1 + were purified by fluorescenceactivated cell sorting from peripheral blood apheresis products of multiple myeloma patients mobilized with cytoxan and GM-CSF. CD34 + Thy-1 + cells were plated at limiting dilution into microtiter wells and cultured in an Iscove's based serumdeprived culture medium, supplemented with the cytokines, interleukin (IL)-3, IL-6, G-CSF, Flk2/Flt3 ligand (FL) and Kit ligand (KL). After 2 weeks, cell proliferation in individual wells was quantified by microscopy and bright-field imaging, or by using a fluorescent nucleic acid-binding dye and fluorimetry. Poisson statistics were used to calculate the frequency of wells containing cells with high proliferative potential (wells containing ≥500 cells). Results Progenitor cell frequencies generated using this assay were compared by linear regression analysis to those generated from 32 parallel CAFC and CFU-C assays performed on the same patient samples. Correlations were r=0.80, r 2 =0.65, and r=0.76, r 2 =0.58, respectively; these correlations were highly significant (p –7 ). Discussion This limiting dilution assay should more directly quantitate the potential of primitive hematopoietic cells than a CFU-C assay. It also has advantages over both the CAFC and the CFU-C assay, in that scoring has been automated, making it simple, rapid, and objective compared with manual cobblestone area or colony counting. The described limiting dilution assay may provide a useful alternative to assays currently used to evaluate the viability and proliferative potential of purified hematopoietic cells intended for transplant.