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CD34+/CD33- cells reselected from macrophage inflammatory protein 1
alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly
enriched for long-term bone marrow culture initiating cells
CM Verfaillie and JS Miller
Stem Cell Laboratory, University of Minnesota, Minneapolis.
Human hematopoietic stem cells are thought to express the CD34 stem cell
antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment
antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell
sorted CD34+ subpopulations contain not more than 1% to 5% primitive
progenitors capable of initiating and sustaining growth in long-term bone
marrow culture initiating cells (LTBMC-ICs). We have recently shown that
culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal
layer by a microporous membrane ("stroma- noncontact" culture) results in
the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of
CD34+ subpopulations still present after several weeks in stroma-noncontact
cultures may result in the selection of cells more highly enriched for
human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3
weeks in stroma- noncontact cultures. Cultured progeny was then sorted on
the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells
evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We
show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA-
DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/-
0.4%). This suggests that HLA-DR antigen expression may depend on the
activation status of primitive cells rather than their lineage commitment.
We then sorted cultured cells on the basis of the myeloid commitment
antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells
contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells
contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained
significantly better in stroma-noncontact cultures supplemented with
macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3
(IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the
frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3)
CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures
contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in
cultured CD34+ subpopulations may be the result of terminal differentiation
of less primitive progenitors, loss of cells that fail to respond to the
culture conditions or recruitment of quiescent LTBMC-ICs. The capability to
select progenitor populations containing up to 30% LTBMC-ICs should prove
useful in studies examining the growth requirements, self-renewal, and
multilineage differentiation capacity of human hematopoietic stem cells at
the single-cell level.
Volume 84,
Issue 5,
pp. 1442-1449,
09/01/1994
Copyright © 1994 by The American Society of Hematology
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