Quantitative Long-Term Culture-Initiating Cell Assays Require Accessory Cell Depletion That Can Be Achieved by CD34-Enrichment or 5-Fluorouracil Exposure

Abstract

Characterization of hematopoietic cells and measurement of their proliferative potential is critical in many research and clinical applications. Because in vivo assay of human cells is not possible and xenogeneic assays are not yet routine, in vitro assays such as the long-term culture-initiating cell (LTC-IC) assay have been widely adopted. This study investigated LTC-IC assay linearity and reproducibility and resulting implications with respect to quantitation of primitive cell expansion. Measurement of secondary colony-forming cells (2° CFCs) from 5-week cultures of bone marrow (BM) mononuclear cells (MNCs) showed that 2° CFC frequency varied with assay plating density in a nonlinear fashion. The measured 2° CFC frequency increased from 4.6 to 63.8 (per 105 MNCs) as assay plating density was decreased from 5 × 105 to 2 × 104 MNCs per well (P < 10−6, n = 37). In contrast, assay of CD34-enriched cells was linear within the range studied. Assays of cells obtained from expansion cultures initiated with either MNCs or CD34-enriched cells were also nonlinear. Consequently, calculated 2° CFC expansion ratios were ambiguous and dependent on the assay plating densities used. Limiting dilution analysis (LDA) results were also nonlinear, with LTC-IC frequency increasing from 8.2 to 22.4 per 105 MNCs (P < 10−4, n = 100) as assay plating densities were decreased. Despite the nonlinearity, 2° CFC and LTC-IC assay results were consistent and reproducible over time with different samples and techniques and gave a semiquantitative indication of relative primitive cell frequency. Although CD34-enriched cells gave linear assay output, purification of cells for every assay is impractical. Therefore, exposure of cells to 5-fluorouracil (5-FU) was explored for improving assay linearity. Incubation of MNCs in 250 μg/mL 5-FU for 1 to 2 hours depleted accessory cells and resulted in a cell population that gave linear 2° CFC readout. The 5-FU–resistant LTC-ICs accounted for 49% of the total LTC-IC population, adding the potential benefit of restricting assay measurement to more primitive noncycling LTC-ICs. Consequently, similar linear assay results can be obtained with either the bulk 2° CFC or LDA LTC-IC methods after 5-FU, but multiple plating densities are nevertheless still required in both methods due to the greater than 100-fold range in primitive cell frequency present in normal human donor BM.