Generation of osteoclasts from isolated hematopoietic progenitor cells

Abstract

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.

The role of IL‐3 in bone

“…22 Using 5-fluorouracil (5-FU)-treated murine spleen cells Kurihara et al showed that TRAP + multinucleated cells developed in the presence of recombinant IL-3 and l,25(OH)2D3 or parathyroid hormone (PTH). 48 In another study, Myint et al showed that IL-3 can induce OC formation in osteopetrotic op/op mice which lacked M-CSF. The subcutaneous injection of 5 ng of rmGM-CSF, 100 ng of rmIL-3, or both, daily for 2 weeks into young op/op mice increased macrophages as well as TRAP + cells in BM.…”

“…Hattersley et al showed that IL‐3 itself induces differentiation of murine BM cells into OCs and their bone resorption activity in vitro in the presence of 1,25‐dihydroxyvitamin D3 . Using 5‐fluorouracil (5‐FU)‐treated murine spleen cells Kurihara et al showed that TRAP + multinucleated cells developed in the presence of recombinant IL‐3 and l,25(OH)2D3 or parathyroid hormone (PTH) . In another study, Myint et al showed that IL‐3 can induce OC formation in osteopetrotic op/op mice which lacked M‐CSF.…”

Human osteopetroses and the osteoclast V-H+-ATPase acidification system

“…Skeletal homeostasis, including normal bone density, is determined by a balance between matrix formation and mineralization functions of osteoblasts, and the resorption of mineralized bone by osteoclasts. It is now well established that osteoclasts originate from pluripotent hematopoietic stem cells, from which other leucocytes are derived, by the fusion of mononuclear cell precursors to form multinucleated giant cells specializing in lacuna resorption (1)(2)(3). Walker first advanced evidence supporting this conclusion following successive experiments with parabiosis marrow transplant (4;5).…”