| A variety of studies has shown that osteoclasts originate from highly enriched hematopoietic stem cells. An enrichment for primitive hematopoietic progenitorscan be derived in mice by the treatment with a high dose of 5-fluorouracil (5-FU) ;Using this technique , a class of murine hematopoietic colonies was prepared that had very high secondary plating efficiencies in vitro. When spleen pluripotent stem cells from mice pretreated in vivo with 5-FU were cultured in the medium containing recombinant interleukin-3 -, 6 (rIL-3-. 6), small colonies ,which consist of blast cells with little sign of differentiation, developed on the day 7 of culture. These blast colonies were lifted, pooled and replated in the presence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) (200U/ml). The blast cells were replated into microtiter wells and cultured with 1 a , 25-(OH)2D3 (10"8mol/L). On the day 21 of culture, appeared multinucleated cells with some characteristics of osteoclasts, such as tartrate-resistant acid phosphatase (TRAP) activity. Moreover, it was shown by electron microscopy that these cells presented resorbptive activity on bone slice and excavations were then formed. This experimental model has become a useful tool for further study of osteoclast formation in vitro.hi summary, rlL- 6-supported blast cell line was prepared, derived from spleen pluripotential stem cells which originally obtained from 5-FU-pretreated mice. Multinucleated cells were induced by GM-CSF and 1 a , 25- (OH) 23 with characteristics of osteoclasts from these blast cells.
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