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Long-term culture-initiating cell expansion is dependent on frequent medium
exchange combined with stromal and other accessory cell effects
MR Koller, MA Palsson, I Manchel and BO Palsson
Aastrom Biosciences, Inc, Ann Arbor, MI 48106, USA.
Despite considerable effort, the expansion of long-term culture- initiating
cells (LTC-ICs) in cultures of purified hematopoietic cells has not yet
been achieved. In contrast, LTC-IC expansion has been attained in cultures
of bone marrow mononuclear cells (MNC) using frequent medium exchange. The
use of frequent medium exchange was, therefore, examined in cultures of
CD34-enriched cells. In stromal- free, CD34-enriched cell cultures, medium
exchange intervals ranging from 2 days to no feeding for 14 days gave
similar results. Six different growth factor combinations, reported by
other groups to give optimal expansion of CD34-enriched cells, were tested
in comparison with the control combination of IL-3/GM-CSF/Epo/SCF. None of
the combinations resulted in improved colony-forming unit-granulocyte
macrophage (CFU-GM) expansion or LTC-IC maintenance, although two were
equivalent. All stromal-free cultures resulted in loss of LTC-IC to half of
input. Because of the limited effect of medium exchange and growth factor
variations on CD34-enriched cell cultures, the effect of preformed stroma
was next examined. Preformed stroma increased cell (3- fold), CFU-GM
(5-fold), and LTC-IC (3-fold) output, but only when the medium was
exchanged every other day. Under these conditions, the number of LTC-IC was
maintained near input level. The lack of LTC-IC expansion in CD34-enriched
cell cultures prompted experiments to examine the effect of cell
purification. Parallel cultures were performed at CD34+lin- cell purities
of 20%, 40%, 70%, and 95%, with each well containing exactly 4,000
CD34+lin- cells in addition to the CD34- accessory cells required to give
the desired percentage. Also, MNC from the same source (approximately 2%
CD34+lin-) were cultured at a concentration to give 4,000 CD34+lin- cells
per well. As CD34+lin- cell purity was decreased from 95% to 2%, the output
of cells, CFU-GM, and LTC-IC increased by threefold to fivefold. The loss
of culture performance with purification was likely due to the removal of
important accessory cells, because the levels of endogenously produced
leukemia inhibitory factor and IL-6 were found to decline significantly
with increasing CD34+lin- cell purity. In summary, preformed stroma
abrogated the decrease in cell and CFU-GM output from cultured CD34-
enriched cells and led to LTC-IC maintenance. In contrast, MNC inocula
resulting in a growing stromal layer during the culture led to LTC-IC
expansion (3.2-fold).(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 86,
Issue 5,
pp. 1784-1793,
09/01/1995
Copyright © 1995 by The American Society of Hematology
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