Abstract
BackgroundBone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells. Many of the details of osteoclast formation, activation and motility remain unsolved. Therefore, there is an interest among bone biologists to transfect the terminally differentiated osteoclasts and follow their responses to the transgenes in vitro. Severe difficulties in transfecting the large, adherent osteoclasts have been encountered, however, making the use of modern cell biology tools in osteoclast research challenging. Transfection of mature osteoclasts by non-viral gene transfer systems has not been reported.ResultsWe have systematically screened the usefulness of several commercial DNA transfection systems in human osteoclasts and their mononuclear precursor cell cultures, and compared transfection efficacy to adenoviral DNA transfection. None of the liposome-based or endosome disruption-inducing systems could induce EGFP-actin expression in terminally differentiated osteoclasts. Instead, a massive cell death by apoptosis was found with all concentrations and liposome/DNA-ratios tested. Best transfection efficiencies were obtained by adenoviral gene delivery. Marginal DNA transfection was obtained by just adding the DNA to the cell culture medium. When bone marrow-derived CD34-positive precursor cells were transfected, some GFP-expression was found at the latest 24 h after transfection. Large numbers of apoptotic cells were found and those cells that remained alive, failed to form osteoclasts when cultured in the presence of RANKL and M-CSF, key regulators of osteoclast formation. In comparison, adenoviral gene delivery resulted in the transfection of CD34-positive cells that remained GFP-positive for up to 5 days and allowed osteoclast formation.ConclusionOsteoclasts and their precursors are sensitive to liposomal transfection systems, which induce osteoclast apoptosis. Gene transfer to mononuclear osteoclast precursors or differentiated osteoclasts was not possible with any of the commercial transfection systems tested. Osteoclasts are non-dividing, adherent cells that are difficult to grow as confluent cultures, which may explain problems with transfection reagents. Large numbers of αvβ3 integrin on the osteoclast surface allows adenovirus endocytosis and infection proceeds in dividing and non-dividing cells efficiently. Viral gene delivery is therefore currently the method of choice for osteoclast transfection.
Highlights
Bone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells
Multinuclear osteoclasts are formed by fusion of their committed mononuclear precursor cells and RANKL is the major growth factor inducing osteoclast formation [5]
Cells were treated with transfection reagents for 2 h, followed by culture for 4 h, 8, or 24 h
Summary
Bone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells. Osteoclasts are bone-resorbing cells that are highly polarized when physiologically active [1]. Their mononuclear precursors are hematopoietic in origin, and remain nonadherent in culture until they differentiate further from the multipotent cell lineage [2,3]. Multinuclear osteoclasts are formed by fusion of their committed mononuclear precursor cells and RANKL is the major growth factor inducing osteoclast formation [5]. Osteoclast morphology and activity is highly dependent on the matrix that they are cultured on, bone being their natural substrate. Mature osteoclasts undergo several cycles of activation and inactivation, where bone is resorbed in the active state and cells migrate in the resting state. The cells die apoptotically and, in vivo, new bone formation by osteoblastic cells takes place to fill the resorption lacuna
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