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Generation of osteoclasts from isolated hematopoietic progenitor cells
N Kurihara, T Suda, Y Miura, H Nakauchi, H Kodama, K Hiura, Y Hakeda and M Kumegawa
Department of Periodontology, Meikai University School of Dentistry, Japan.
A variety of studies have shown that osteoclasts originate from bone
marrow, but their exact progenitors and differentiation pathway remain
unclear. The treatment of mice with a high dose of 5-fluorouracil (5- FU)
results in an enrichment for primitive hematopoietic progenitors; using
this procedure, we prepared a new class of murine hematopoietic colonies
that had very high secondary plating efficiencies in vitro. When spleen
cells from mice pretreated in vivo with 5-FU were cultured in the presence
of methylcellulose medium containing recombinant interleukin-3 (rIL-3),
small colonies consisting of blast cells with little sign of
differentiation developed on day 7 of culture. We lifted these blast
colonies, pooled them, and replated them as secondary methylcellulose
cultures in the presence of rIL-3 and erythropoietin. Approximately 60% of
the cells formed colonies comprising various combinations of neutrophils,
macrophages, eosinophils, mast cells, megakaryocytes, and erythroblasts. We
replated such blast cells into microtiter wells and cultured them in the
presence of rIL-3 (100 U/mL) or recombinant granulocyte-macrophage colony
stimulating factor (GM- CSF) (100 U/mL) plus 1.25(OH)2D3 (10(-7) mol/L).
Multinucleated cells appeared from day 14 of culture and approximately 100
giant cells per well were scored on day 21 of culture. Parathyroid hormone
(1 U/mL) also induced the multinucleated cell formation.
May-Grunwald-Giemsa staining revealed the large cells containing many
nuclei in their cytoplasm, which is characteristic of bone-resorbing cells
or osteoclasts. These cells showed a tartrate-resistant acid phosphatase
(TRAP) activity. Calcitonin caused a striking shape change in these cells
and suppressed the formation of multinucleated cells. Moreover, electron
microscopy shows that these cells were able to resorb fetal calvariae. In
the presence of r granulocyte-colony stimulating factor, r
macrophage-colony stimulating factor, or r interleukin-6 plus 1.25(OH)2D3,
formation of TRAP-positive multinucleated cells was lower compared with the
support of rIL-3 or rGM-CSF. Mature macrophages collected from colonies did
not form the multinucleated cells as described above, even in the presence
of rIL-3 and 1.25(OH)2D3. Moreover, to exclude the possibility that
osteoclasts generated from non-blast cells, we performed a cloning
experiment from one isolated blast cell and demonstrated that single cells
differentiate into osteoclasts or macrophages in the presence of rIL-3 with
or without 1.25(OH)2D3. This system will provide a useful model for further
analysis of osteoclast formation in vitro.
Volume 74,
Issue 4,
pp. 1295-1302,
09/01/1989
Copyright © 1989 by The American Society of Hematology
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