Isolation and characterization of highly enriched, prefusion mouse osteoclastic cells.
This study describes the isolation and characterization of highly enriched mammalian osteoclast precursors, released by the "disintegrin" echistatin, from an osteoclast formation culture. Incubation of a 6-day coculture of mouse bone marrow cells and mouse osteoblastic cells (MB1.8) with echistatin (30 nM), an RGD-containing snake venom, for 20 min yielded an 88-95% pure population of tartrate-resistant acid phosphatase-positive cells, 1.5 x 10(5) cells per 150 cm2 culture dish. These cells were mostly mononucleated and based on the following characteristics are considered to be prefusion osteoclasts (pOC cells): (i) presence of calcitonin (CT) receptors documented by 125I-sCT autoradiography and cAMP generation in response to salmon calcitonin; (ii) expression of mRNAs for alpha v beta 3 integrin, osteopontin, 92-kDa type IV collagenase (matrix metalloproteinase 9), carbonic anhydrase II, OC-2 (an "osteoclastic" cysteine proteinase), and protein tyrosine phosphatase epsilon; and (iii) high level expression of pp60c-src protein. The pOC cells resorb bone (form "pits" on bone slices) but only in the presence of osteoblastic MB1.8 cells and 1,25(OH)2D3. Resorption was inhibited by CT. In conclusion, we describe a rapid, reproducible procedure to isolate virtually pure mammalian prefusion osteoclasts, which should help in the study of osteoclast formation, composition, and function.
Wesolowski
G
G
Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Duong
L T
LT
Lakkakorpi
P T
PT
Nagy
R M
RM
Tezuka
K
K
Tanaka
H
H
Rodan
G A
GA
Rodan
S B
SB
D000818:Animals; D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D016023:Integrins / Q000096:biosynthesis; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D053495:Osteopontin; D010455:Peptides / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000096:biosynthesis; D014757:Viper Venoms / Q000494:pharmacology
D016023:Integrins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D014757:Viper Venoms; D053495:Osteopontin; C057986:echistatin