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Quantitation of the quiescent fraction of long-term culture-initiating
cells in normal human blood and marrow and the kinetics of their growth
factor-stimulated entry into S-phase in vitro
L Ponchio, E Conneally and C Eaves
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
A method for quantitating the proportion of cycling long-term culture-
initiating cells (LTC-IC) in heterogeneous populations of human
hematopoietic cells is described. This procedure involves incubating the
cells of interest for 16 to 24 hours in a serum-free medium containing 100
ng/mL Steel factor (SF), 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL
granulocyte-colony-stimulating factor (G-CSF), with or without 20
microCi/mL of high specific activity 3H-thymidine (3H-Tdr) before plating
the recovered cells in standard LTC-IC assays. The details of this
procedure are based in part on the finding that the number of LTC-IC
(regardless of their cycling status) remains constant for at least 24 hours
under these culture conditions, as long as 3H-Tdr is not present. In
addition, we have determined that a 16-hour period of exposure to the
3H-Tdr is sufficient to maximize the discrimination of cycling LTC-IC but
not long enough to allow a detectable redistribution of LTC-IC between
noncycling and cycling compartments. Finally, any isotope reutilization
that may occur is not sufficient to affect the LTC-IC 3H-Tdr suicide values
measured. Application of this methodology to normally circulating LTC-IC
showed these to be a primarily quiescent population. However, within 72
hours of incubation in a serum-free medium containing SF, IL-3, and G-CSF,
most had entered S-phase, although there was no net change in their
numbers. This suggests that, under certain conditions in vitro,
self-renewal divisions of LTC-IC can occur and, at least initially, balance
any losses of these cells due to their differentiation or death. In
contrast, many of the LTC-IC in freshly aspirated samples of normal marrow
were found to be proliferating, although those that were initially
quiescent could also be recruited into S-phase within 72 hours in vitro
when incubated under the same conditions used to stimulate circulating
LTC-IC. This modified 3H-Tdr suicide procedure should facilitate further
investigation of the mechanisms regulating the turnover of the most
primitive compartments of human hematopoietic cells and how these may be
altered in disease states or exploited for a variety of therapeutic
applications.
Volume 86,
Issue 9,
pp. 3314-3321,
11/01/1995
Copyright © 1995 by The American Society of Hematology
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