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Isolation in a single step of a highly enriched murine hematopoietic stem
cell population with competitive long-term repopulating ability
SJ Szilvassy, PM Lansdorp, RK Humphries, AC Eaves and CJ Eaves
Terry Fox Laboratory, B. C. Cancer Research Centre, Vancouver.
A simple procedure is described for the quantitation and enrichment of
murine hematopoietic cells with the capacity for long-term repopulation of
lymphoid and myeloid tissues in lethally irradiated mice. To ensure
detection of the most primitive marrow cells with this potential, we used a
competitive assay in which female recipients were injected with male "test"
cells and 1 to 2 x 10(5) "compromised" female marrow cells with normal
short-term repopulating ability, but whose long-term repopulating ability
had been reduced by serial transplantation. Primitive hematopoietic cells
were purified by flow cytometry and sorting based on their forward and
orthogonal light-scattering properties, and Thy-1 and H-2K antigen
expression. Enrichment profiles for normal marrow, and marrow of mice
injected with 5-fluorouracil (5- FU) four days previously, were established
for each of these parameters using an in vitro assay for high proliferative
potential, pluripotent colony-forming cells. When all four parameters were
gated simultaneously, these clonogenic cells were enriched 100-fold. Both
day 9 and day 12 CFU-S were copurified; however, the purity (23%) and
enrichment (75-fold) of day 12 CFU-S in the sorted population was greater
with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells
consistently repopulated recipient lymphoid and myeloid tissues (greater
than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2
x 10(5) compromised female marrow cells, and approximately 100 were
sufficient to achieve the same result in 50% of recipients under the same
conditions. This relatively simple purification and assay strategy should
facilitate further analysis of the heterogeneity and regulation of stem
cells that maintain hematopoiesis in vivo.
Volume 74,
Issue 3,
pp. 930-939,
08/15/1989
Copyright © 1989 by The American Society of Hematology
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