Quantitative Long-Term Culture-Initiating Cell Assays Require
Accessory Cell Depletion That Can Be Achieved by
CD34-Enrichment or 5-Fluorouracil Exposure
Manfred R. Koller,
Ilana Manchel, and
Alan K. Smith
From Aastrom Biosciences, Inc, Ann Arbor, MI.
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 < 104, 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.
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4056-4064
© 1998 by The American Society of Hematology.
