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Retention of quiescent hematopoietic cells with high proliferative
potential during ex vivo stem cell culture
JC Young, A Varma, D DiGiusto and MP Backer
SyStemix Inc, Department of Cell Product Development, Palo Alto, CA 94304,
USA.
Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM)
or mobilized peripheral blood (MPB) are highly enriched for pluripotent
stem cells. Ex vivo expansion of this population is proposed as a means of
providing accelerated short-term, as well as long-term, engraftment after
myeloablative therapy. Here we demonstrate that primitive quiescent cells
are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that
the cell production capacity of the expanded cell product can largely be
attributed to cells exhibiting quiescent behavior during culture.
CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the
fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells
on a murine stromal layer in the presence of interleukin (IL)-3, IL-6,
c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each
subsequent cell division, PKH26 fluorescence is reduced by roughly half,
which allows tracking of the number of cell divisions. Progenitor cells
present after a 2-week expansion period were sorted into
CD34+/Lin-/dyebright and CD34+/Lin- /dyedim fractions and then cultured in
a 4-week single-cell proliferation assay to characterize the proliferative
capacity of each group. Fifty-nine percent of progenitors remaining
dyebright after bulk culture (four or fewer cell divisions) were observed
to proliferate in single cell culture, and produced an average of 1,780
cells per plated cell. In contrast, only 26% of dyedim (more than four
divisions) progenitors were observed to proliferate and displayed a lower
average proliferative capacity of 225 cells per plated cell. Similar
behaviors were observed after a second consecutive cycle of bulk culture,
indicating that quiescent cells with high proliferative capacity existed in
culture for at least 4 weeks. Single CD34+/Lin-/dyebright progenitors
purified from bulk cultures were observed to produce as many as 1,000 CD34
positive progeny during single cell culture, and these progeny included
multilineage colony forming cells. These data demonstrate that among CD34
positive cells recovered after in vitro bulk culture, a higher
proliferative capacity correlated with quiescent behavior. The described
culture method provides quantitation of the cell producing capacity of
individual cells in hematopoietic cell mixtures and may prove useful for
predicting engrafting potential in products intended for cellular therapy.
Volume 87,
Issue 2,
pp. 545-556,
01/15/1996
Copyright © 1996 by The American Society of Hematology
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