Abstract
In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or genetically manipulated mouse lines. These cells represent a standardized and theoretically unlimited source for osteoclast-associated research projects.
Highlights
Homeostasis and controlled remodeling of bone tissues are maintained by the coupled and balanced action of bone resorbing osteoclasts (OCs) and bone forming osteoblasts [1,2,3]
Familial Paget’s disease of bone (PDB) is associated with mutations in the ubiquitin associated (UBA) domain of sequestosome 1 which encodes p62, a scaffold protein known to be involved in cytokine signaling, and that can serve as a cargo adaptor for polyubiquitinated proteins [4]
Our study describes the differentiation and functional characterization of OCs from immortalized myeloid progenitor cells that were isolated from BM of different wild type (WT) and genetically modified mice
Summary
Homeostasis and controlled remodeling of bone tissues are maintained by the coupled and balanced action of bone resorbing osteoclasts (OCs) and bone forming osteoblasts [1,2,3]. Mature OCs can be identified by different biological markers such as tartrate-resistant acid phosphatase (TRAP) staining, multi-nucleation, F-actin ring formation and their unique bone resorbing capacity [10,11]. Besides their primary function in the regulation of bone resorption, OCs are important orchestrators of several other processes, e.g. the regulation of hematopoiesis, bone formation and angiogenesis of blood vessels during bone development [12,13]
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