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Identification of hematopoietic stem cell subsets on the basis of their
primitiveness using antibody ER-MP12
JC van der Loo, WA Slieker, D Kieboom and RE Ploemacher
Institute of Hematology, Erasmus University, Rotterdam, The Netherlands.
Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic
stem cells. The antigen is differentially expressed by different subsets in
the hematopoietic stem cell compartment and enables a physical separation
of primitive long-term repopulating stem cells from more mature
multilineage progenitors. When used in two-color immunofluorescence with
ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow (BM) cells could be
identified. These subsets were isolated using magnetic and
fluorescence-activated cell sorting, phenotypically analyzed, and tested in
vitro for cobblestone area-forming cells (CAFC) and colony-forming units in
culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for
day-12 spleen colony-forming units (CFU-S-12), and for cells with long-term
repopulating ability using a recently developed alpha-thalassemic chimeric
mouse model. Cells with long-term repopulation ability (LTRA) and day-12
spleen colony-forming ability appeared to be exclusively present in the two
subpopulations that expressed the ER-MP12 cell surface antigen at either an
intermediate or high level, but lacked the expression of Ly- 6C. The
ER-MP12med20- subpopulation (comprising 30% of the BM cells, including all
lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28
CAFC (CAFC-28), 75% of the CFU-S-12, and very low numbers of CFU-C. In
contrast, the ER-MP12hi20- population (comprising 1% to 2% of the BM cells,
containing no mature cells) included 80% of the early and less primitive
CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and
immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen
expression, included 80% to 90% of the CFU-C (day 4 through day 14), of
which 70% were ER-MP20- and 10% to 20% ER-MP20med/hi. In addition,
erythroblasts, granulocytes, lymphocytes, and monocytes could almost be
fully separated on the basis of ER-MP12 and ER-MP20 antigen expression.
Functionally, the presence of ER-MP12 in a long-term BM culture did not
affect hematopoiesis, as was measured in the CAFC assay. Our data
demonstrate that the ER-MP12 antigen is intermediately expressed on the
long-term repopulating hematopoietic stem cell. Its level of expression
increases on maturation towards CFU-C, to disappear from mature
hematopoietic cells, except from B and T lymphocytes.
Volume 85,
Issue 4,
pp. 952-962,
02/15/1995
Copyright © 1995 by The American Society of Hematology
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