Cell Lines to Study Bone Formation and Regulation

University of Missouri System: University of Missouri-Kansas City
posted on 02/01/2010

Novel cell lines that are useful in the examination of osteocyte function, biomineralization, SOST/sclerostin, FGF23 and other mechanisms of osteoblast-to-osteocyte differentiation.

Suggested Uses

To generate large numbers of osteocyte-like cells in order to produce sufficient quantities of osteocytes for study.
To generate large numbers of cells of a homogeneous stage of osteogenic differentiation.
To study osteocyte secretion of sclerostin, such as screening for sclerostin antagonists.
To investigate regulation of FGF23 expression in osteocytes and the role of osteocytes in regulation blood calcium/phosphate homeostasis.
To study the role of osteocytes as mechanosensory cells and their role in regulating bone response to mechanical stress.
To screen potential new therapies to induce bone formation.
To track cells responsible for bone formation in vivo.
To identify additional osteocyte-selective markers and receptors.

Advantages

This invention is an improvement over previous cell lines due to the following factors:

The cells are maintained in a non-differentiated state at 33°C in the presence of interferon- g (IFN-g), which allows large scale production without the loss of phenotype as occurs with other cell lines;
Upon culture at 37°C in the absence of IFN-g, the temperature-sensitive large T-antigen is no longer expressed, no longer functional, and no longer contributes to the cell phenotype. Thus, the cells have the same gene expression as primary cells;
The cells are clonal, so all cells are homogeneous and at the same stage of differentiation;
The IDG-SW3 cells express the series of markers of the early-to-late osteocyte including Dmp1-GFP, E11/gp38, SOST/sclerostin, and FGF23;
These cells can be maintained not only in 2D cultures but also in 3D cultures;
These cells are viable up to 35-50 days; and
These cells will generate new bone in vivo.

Innovation Details

Detailed Description

IDG-SW3 and IDG-T1 represent osteoblastic cell lines with a GFP marker for differentiation that mirror their differentiation into osteocytes and the formation of a mineralized extracellular collagen matrix.

Cell Line Properties:

When cultured at 33°C in the presence of γ-IFN, both cell lines express the SV40 large T antigen and proliferate but do not express Dmp1-GFP.
Under osteogenic culture conditions (50 μg/ml ascorbic acid, 4 mM β-glycerophosphate), both cell lines express the Dmp1-GFP, produce a mineralized extracellular matrix, and no longer express the SV40 large T antigen protein, as determined by Western blot analysis of cell lysates.
Quantitation of GFP expression using relative fluorescence units from total cell lysates show that GFP expression increases steadily from an undetectable baseline at day zero, is first detectable as early as Day 3, increases steadily over time, and is still detectable in cultures at Day 50. GFP expression is uniformly distributed across the culture surface.
Alkaline phosphatase activity increases over time.
Quantitation of Von Kossa and alizarin red staining showed significantly increased mineralization and calcification levels at 7 days, with maximal mineralization at 14 days. GFP-positive cells co-localize with regions of alizarin red staining.
Scanning electron microscopy demonstrates both cell lines produce highly mineralized, dense, extracellular network of collagen fibrils associated with nanospherulites.
Immunofluorescent staining with an antibody specific for type I collagen demonstrates induction of type I collagen expression in culture over time.
Both cell lines express osteocyte markers in osteogenic culture over time.
Immunofluorescent staining with an antibody specific for E11, an early osteocyte marker, showed low baseline E11 expression increasing over time in osteogenic culture conditions.
Western blot analysis demonstrates significant E11 protein induction by Day 7. GFP and E11 expression appeared to co-localize with mineralized areas.
PCR and semi-quantitative real-time PCR of cDNA samples from both cell lines demonstrate induction of Dmp1 and SOST mRNA expression from almost undetectable levels at Day 0 to abundant expression by Day 14 of culture in osteogenic conditions.
Both cell lines are capable of growing on three-dimensional collagen matrices. When cultured on either a 3-dimensional type I collagen gel or type I collagen sponge, both cell lines express Dmp1-GFP, and cells on the surface form multiple layers, with sub-populations of cells becoming embedded at various depths within the collagen and separated from a surface cells by a layer of extracellular matrix.

File Number: 10UMK007

Web site: http://www.umkc.edu/ors/tt/

IP Protection

License Online

This innovation currently is not available for online licensing. Please contact James Brazeal at University of Missouri System: University of Missouri-Kansas City for more information.

request more info