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Phenotypic and functional characterization of competitive long-term
repopulating hematopoietic stem cells enriched from 5-fluorouracil- treated
murine marrow
SJ Szilvassy and S Cory
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne
Hospital, Victoria, Australia.
Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with
5-fluorouracil (5-FU) were characterized with respect to 12 parameters
using fluorescence-activated cell sorting and a competitive long-term
repopulation assay. Stem cells were larger than lymphocytes and exhibited
side light-scatter characteristic of blast cells. Most expressed low levels
of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and
stained brightly with rhodamine-123. Significantly, most long-term
repopulating cells also expressed CD4, some at high density. In addition, a
significant proportion displayed low to medium levels of the
"lineage-specific" markers CD45R (B220), Gr- 1, and TER-119. A simple and
rapid multiparameter sorting procedure enriched the stem cells 100-fold and
substantially removed most other clonogenic cell types, including day 12
spleen colony-forming cells. Cells able to generate cobblestone colonies on
stromal cells in vitro were co-enriched. Lethally irradiated mice
transplanted with limiting numbers of the sorted stem cells did not survive
unless cotransplanted with "compromised" marrow cells prepared by prior
serial transplantation and shown to be depleted of long-term repopulating
activity. A significant number of recipients transplanted with 25 to 100
sorted cells contained donor-derived B and T lymphocytes and granulocytes
in their peripheral blood for at least 6 months. Limiting dilution analysis
in vivo indicated that the frequency of competitive long-term repopulating
units (CRU) in the sorted population was at least 1 in 60 cells. The
calculated frequency of CRU was largely independent of the time of
recipient analysis between 10 and 52 weeks, indicating that highly enriched
stem cells can be recruited relatively early in certain transplant
settings. This simple enrichment and assay strategy for repopulating
hematopoietic stem cells should facilitate further analysis of their
regulation in vivo.
Volume 81,
Issue 9,
pp. 2310-2320,
05/01/1993
Copyright © 1993 by The American Society of Hematology
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