In vitro osteoclast generation from different bone marrow fractions, including a highly enriched haematopoietic stem cell population

It is well established that the osteoclast is formed by fusion of post-mitotic, mononuclear precursors derived from circulating progenitor cells. However, the precise haematopoietic origin of the osteoclast is unknown. We have investigated this here by fractionating mouse bone marrow and isolating haematopoietic stem cells using a three-step method combining equilibrium density centrifugation and two fluorescence-activated cell sortings (FACS), and have tested the ability of each bone marrow fraction, including highly purified haematopoietic stem cells, to generate osteoclasts during co-culture with preosteoclast-free embryonic long bones. The osteoclast-forming capacity was found to increase with increasing stem cell purity. On the other hand, the culture time needed for osteoclast formation also increased with purification, suggesting the presence of progressively more immature progenitor cells. The pluripotent haematopoietic stem cell fractions with the highest purity needed preincubation with a stem cell-activating factor (interleukin-3) to activate the predominantly quiescent stem cells in vitro.