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Characterization and purification of osteogenic cells from murine bone
marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and
wheat germ agglutinin
P Van Vlasselaer, N Falla, H Snoeck and E Mathieu
Department of the Environment, Vlaamse Instelling voor Technologisch
Onderzoek, Mol, Antwerpen, Belgium.
Osteogenic cells were sorted from bone marrow of 5-fluorouracil (5-FU)-
treated mice based on light scatter characteristics, Sca-1 expression, and
their binding to wheat germ agglutinin (WGA). Four sort gates were
established using forward (FSC) and perpendicular (SSC) light scatter and
were denominated as FSChigh SSClow, FSClow SSChigh, FSClow SSClow, and
FSChigh SSChigh cell. Cells from the FSChigh SSChigh gate, but not from the
other gates, synthesized alkaline phosphatase, collagen, and osteocalcin
and formed a mineralized matrix in culture. The number of osteoprogenitor
cells was significantly enriched after depleting the 5- FU bone marrow from
cells of the lymphoid and myeloid lineage, eg, T cells, B cells, natural
killer cells, granulocytes, macrophages, and erythrocytes. Approximately
95% of the FSChigh SSChigh cell population of this "lineage-negative"
(Lin-) marrow expressed the Sca-1 antigen (Sca-1+) and bound WGA. Three
additional sort windows were established based on WGA binding intensity and
were denominated as Sca-1+ WGAdull, Sca-1+ WGAmedium, and Sca-1+ WGAbright.
Cells from the Sca-1+ WGAbright gate, but not from the other gates,
synthesized bone proteins and formed a mineralized matrix. However, they
lost this capacity upon subcultivation. Further immunophenotypic
characterization showed that FSChigh SSChigh Lin- Sca-1+ WGAbright cells
expressed stromal (KM16) and endothelial (Sab-1 and Sab-2) markers, but not
hematopoietic surface markers such as c-kit and Thy1.2. Sorted FSChigh
SSChigh Lin- Sca-1+ WGAbright cells form three-dimensional nodules that
stain with the von Kossa technique and contain osteoblast and
osteocyte-like cells.
Volume 84,
Issue 3,
pp. 753-763,
08/01/1994
Copyright © 1994 by The American Society of Hematology
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