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A novel single-cell proliferation assay shows that long-term culture-
initiating cell (LTC-IC) maintenance over time results from the extensive
proliferation of a small fraction of LTC-IC
CM Verfaillie and JS Miller
Division of Hematology, University of Minnesota, Minneapolis 55455, USA.
We have previously shown that when adult marrow CD34+/HLA-DR- cells are
cultured for 5 or 8 weeks in the presence of stroma-conditioned media with
interleukin-3 (IL-3) and macrophage inflammatory protein-1 alpha (MIP-1
alpha), long-term culture-initiating cells (LTC-IC) are maintained but not
expanded. However, if the same cultures are evaluated after 2 weeks, we
show that LTC-IC expand 5.5- +/- 0.2-fold. Because expansion of LTC-IC is
likely the result of a balance between proliferation and loss of LTC-IC, we
hypothesized that, although LTC-IC proliferate in these cultures, loss of a
fraction of LTC-IC underlies the lack of long-term expansion. To evaluate
the fate of LTC-IC (proliferation, conservation, or loss), we performed
PKH-26 labeling assays and developed a single LTC-IC proliferation assay.
For PKH-26 labeling assays, CD34+/HLA-DR- cells were incubated with the
membrane intercallating dye, PKH-26, before culture for 14 days in stroma-
noncontact cultures + IL-3 + MIP-1 alpha. Progeny was reselected by
fluorescence-activated cell sorting based on their PKH-26 fluorescence
intensity. These studies showed that LTC-IC proliferate because 80% of
LTC-IC at week 2 had 0.5 to 1 log lower fluorescence intensity than did
freshly labeled CD34+/HLA-DR- cells. To further determine the fate of
LTC-IC, we also developed a single LTC-IC proliferation assay. A population
of CD34+/CD33- cells, highly enriched in LTC-IC, was sorted singly in
stroma-conditioned media+IL-3 + MIP-1 alpha. After 5 weeks, the content of
each well was divided equally over 8 secondary stroma- containing wells and
cultured for 8 weeks to determine the capacity of the single-cell progeny
to initiate 1 or more secondary stromal cultures. Progeny of single-sorted
cells were able to initiate up to 8 secondary long-term cultures,
demonstrating that LTC-IC proliferate in stroma-conditioned media+IL-3 +
MIP-1 alpha. However, more than 65% of single-sorted LTC-IC were not
conserved because their progeny could no longer initiate secondary
long-term cultures. This finding indicates that, although stromal factors
and IL-3 + MIP-1 alpha can induce proliferation of LTC-IC, failure to
conserve a large fraction of LTC-IC results in lack of long-term
expansion.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 86,
Issue 6,
pp. 2137-2145,
09/15/1995
Copyright © 1995 by The American Society of Hematology
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