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An in vitro limiting-dilution assay of long-term repopulating hematopoietic
stem cells in the mouse
RE Ploemacher, JP van der Sluijs, JS Voerman and NH Brons
Department of Cell Biology II, Erasmus University, Rotterdam, The
Netherlands.
We have developed a limiting-dilution assay of long-term repopulating
hematopoietic stem cells in the mouse using a miniturized stroma- dependent
bone marrow culture assay in vitro. The cells were overlaid on irradiated
stromal layers in microtiter wells in a range of concentrations, and
frequencies of cobblestone area-forming cells (CAFC) were calculated by
employing Poisson statistics. The production of secondary
granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer
of individual wells was correlated with the presence of such cobblestone
areas. CAFC frequencies were determined in bone marrow cell suspensions
that were either enriched for marrow repopulating ability (MRA) in vivo,
while depleted for spleen colony- forming units (CFU-S), or vice versa. The
separation of bone marrow cells (BMC) was either based on centrifugal
elutriation, or monoclonal antibody-mediated magnetic depletion of cells
carrying cell surface differentiation antigens, and subsequent sorting on
the basis of light scatter and rhodamine-123 retention as a measure of
mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were
studied. Our investigations show that a time-dependent cobblestone area
formation exists that reflects the turnover time and primitiveness of CAFC.
The frequency of precursors forming cobblestone areas on day 28 after
overlay is proposed to be a measure for MRA, whereas the day-7 CAFC
frequency closely corresponds with day-12 CFU-S numbers in the suspensions
tested.
Volume 74,
Issue 8,
pp. 2755-2763,
12/01/1989
Copyright © 1989 by The American Society of Hematology
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