A Novel Osteogenic Cell Line That Differentiates Into GFP-Tagged Osteocytes and Forms Mineral With a Bone-Like Lacunocanalicular Structure

Abstract

Osteocytes, the most abundant cells in bone, were once thought to be inactive, but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane-targeted GFP driven by the Dmp1-promoter. One of these clones, OmGFP66, has unique properties compared with previous osteogenic and osteocyte cell models and forms 3-dimensional mineralized bone-like structures, containing highly dendritic GFP-positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone-like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase (ALP), produce a mineralized type I collagen matrix, and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23, and the mature osteocyte marker, Sost. They also express RankL, Opg, and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1, and Opg and upregulation of RankL and E11/gp38. Live cell imaging revealed the dynamic process by which OmGFP66 bone-like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule-like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function, and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment.

Osteocyte apoptosis: the roles and key molecular mechanisms in resorption-related bone diseases

“…Importantly, PTHrP, much like PTH, displays anti-apoptotic features in osteoblasts and osteocytes 132 – 134 . Similarly, it also was corroborated that many of the osteocyte-related genes, such as SOST, DMP-1, were downregulated by PTH in an expected manner in OmGFP66 cells (immortalized osteogenic cell lines) derived from primary osteocytes of transgenic mice (DMP1-promoter) 29 . Based on these findings, it is demonstrated that the PTHrP/PTH1R pathway functions as an essential pathway in preserving the osteocytic vitality upon mechanical stimulus (Fig.…”

“…Trigger the generation of pro-inflammatory and pro-osteoclastic factors via positive feedback loop [109][110][111][112]114 FADD Trigger a caspase cascade; induce GCs-induced apoptosis 6,118 Sclerostin Promote osteocyte cells death upon unloading; inhibit bone formation 53,107,108 BNIP3 Promote cell death during hypoxia 60,62 CCN2 Promote osteocyte apoptosis upon excess mechanical stress 33,69 TNF-α Stimulate osteocyte apoptosis upon inflammation and cancer 92,115,116 Caspase-3 Regulate osteocyte apoptosis via physical interactions in mechanistic stimulus 27,127 CTSK Breakdown the bone matrix adjacent to the osteocyte; increase the size of the osteocyte lacunae and mineralization decrease vitality of osteocytes 23 DMP-1 Regulate osteocyte formation and phosphate homeostasis; involve in osteocytic apoptosis 29,84,154 Pyk2 Promote GCs-induced osteocytic apoptosis via focal adhesion 82 Panx-1 Promote fatigue-induced osteocytic apoptosis 160 Anti-apoptotic function SOD2 Suppress aging and loss of bone mass; decrease degeneration of the osteocyte LCN 24,61 AMPK Protects against Hcy-induced osteocyte apoptosis 96,101,102 NO Maintain osteocytic vitality by pulsatile fluid flow 44,54,100…”

“…Therefore, it might be necessary to utilize novel approaches to elucidate the findings in humans, such as the application of human-derived bone cells and immortalized bone cell lines with CRISPR-Cas9 knockout. Until recently, it has been reported that the establishment of two novel osteogenic cell lines (OmGFP66 and OmGFP10) forms 3D bone-like structures with an extensive interconnected LCN structure that mimics bone in vivo 29 . In addition, a modified method to isolate primary human osteocytes from bone also has been reported to significantly improve the yield of osteocytes 30 .…”

Fibroblast growth factor 9 (FGF9) inhibits myogenic differentiation of C2C12 and human muscle cells

“…The specific role for FGF9 in skeletal muscle differentiation is elusive. Recent studies from our groups showed that FGF9 mRNA expression is highly enriched in osteocytes in the OmGFP66 osteogenic cell line as compared to the osteoblast population [40,41]. We have reasoned that this molecule could be functioning as an osteokine based on our observations (Huang & Brotto, Unpublished Results) that fibroblast-conditioned media can inhibit C2C12 differentiation.…”

A New Osteocytic Cell Line, Raising New Questions and Opportunities

“…The major expectation from these cell lines is that they develop osteocytic‐like cells within a mature bone matrix, form lacunocanalicular structures, and allow 3D organization of osteocytes. In this issue of the Journal of Bone and Mineral Research ( JBMR ), Wang and colleagues report a novel cell line that meets most of these morphological criteria. Two novel cell lines were derived from osteoblast‐rich calvarial digests from mice, using membrane‐bound GFP (AcGFP‐mem; Clontech) driven by the DMP1 promoter as selection mechanism.…”