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Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells.

Zach F, Mueller A, Gessner A - PLoS ONE (2015)

Bottom Line: 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.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.These cells represent a standardized and theoretically unlimited source for osteoclast-associated research projects.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

C3H/HeJ ER-Hoxb8-derived OCs show different stages of F-actin ring formation.Visualization of F-actin ring and podosome structures in permeabilized (Triton™ X-100) OCs was enabled by interaction of FITC-conjugated phalloidin (green) with F-actin. Nuclei were counterstained with DAPI (blue). Representative fluorescence microscopy images of F-actin structures observed in ER-Hoxb8-derived OCs show examples of podosome cluster, podosome ring and podosome belt in cells differentiated on uncoated glass coverslips. A representative example of fluorescence microscopy images of mature F-actin ring structures in OCs which were differentiated on 100 mm dishes (300,000 cells) and subsequently plated on CaP-coated glass coverslips is shown in the lowest panel. Scale bars = 50 μm.
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pone.0142211.g005: C3H/HeJ ER-Hoxb8-derived OCs show different stages of F-actin ring formation.Visualization of F-actin ring and podosome structures in permeabilized (Triton™ X-100) OCs was enabled by interaction of FITC-conjugated phalloidin (green) with F-actin. Nuclei were counterstained with DAPI (blue). Representative fluorescence microscopy images of F-actin structures observed in ER-Hoxb8-derived OCs show examples of podosome cluster, podosome ring and podosome belt in cells differentiated on uncoated glass coverslips. A representative example of fluorescence microscopy images of mature F-actin ring structures in OCs which were differentiated on 100 mm dishes (300,000 cells) and subsequently plated on CaP-coated glass coverslips is shown in the lowest panel. Scale bars = 50 μm.

Mentions: In order to clarify whether ER-Hoxb8-derived OCs may be designated as a new source of bona fide OCs, we examined the functionality of these cells in comparison to OCs derived from conventional OC progenitor sources. Mature and functional OCs are known to develop an F-actin ring, which is important for the formation of a sealing zone that is associated with OC bone resorption activity [11]. Prior to the arrangement of a mature F-actin ring, actin structures of OCs pass through different intermediate developmental stages called podosome cluster, podosome ring and podosome belt [11,37]. To investigate these structures in ER-Hoxb8-derived OCs, cells were subjected to FITC-phalloidin staining at d5 of OC differentiation. Fig 5 illustrates podosome cluster, podosome ring and podosome belt structures in multi-nucleated cells (C3H/HeJ WT) differentiated on glass coverslips. These structures, which were also visible in BALB/c WT and p62 KO ER-Hoxb8-derived OCs (data now shown), were detectable in comparable quantities and qualities when cells were first differentiated and subsequently plated on biomimetic CaP-coated coverslips (data not shown). It is known that F-actin ring structures, which indicate actively resorbing OCs, are optimally visible on dentin or biomimetic bone substrates, for example on CaP-coated cell culture plates or glass coverslips, and thus fully developed F-actin rings are not likely to be detectable on non-coated plates or coverslips. In order to overcome these limitations, mature OCs, initially differentiated from ER-Hoxb8 progenitors in standard cell culture dishes, were isolated and subsequently plated on CaP-coated glass coverslips. Following incubation for 48 h, cells were stained for F-actin with FITC-phalloidin. Fig 5 (lowest panel) shows a representative multi-nucleated OC containing a heavily stained F-actin ring structure on the cell periphery. Cell movement and signs of OC-associated bone resorption activity at the interface of the F-actin ring are depicted in S1 Fig. The overview of merged bright-field, FICT-phalloidin and DAPI images from ER-Hoxb8-derived OCs (C3H/HeJ WT, p62 KO) points to a directional CaP resorption activity, which is clearly linked to and dependent on its F-actin ring formation on the cell periphery.


Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells.

Zach F, Mueller A, Gessner A - PLoS ONE (2015)

C3H/HeJ ER-Hoxb8-derived OCs show different stages of F-actin ring formation.Visualization of F-actin ring and podosome structures in permeabilized (Triton™ X-100) OCs was enabled by interaction of FITC-conjugated phalloidin (green) with F-actin. Nuclei were counterstained with DAPI (blue). Representative fluorescence microscopy images of F-actin structures observed in ER-Hoxb8-derived OCs show examples of podosome cluster, podosome ring and podosome belt in cells differentiated on uncoated glass coverslips. A representative example of fluorescence microscopy images of mature F-actin ring structures in OCs which were differentiated on 100 mm dishes (300,000 cells) and subsequently plated on CaP-coated glass coverslips is shown in the lowest panel. Scale bars = 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4631598&req=5

pone.0142211.g005: C3H/HeJ ER-Hoxb8-derived OCs show different stages of F-actin ring formation.Visualization of F-actin ring and podosome structures in permeabilized (Triton™ X-100) OCs was enabled by interaction of FITC-conjugated phalloidin (green) with F-actin. Nuclei were counterstained with DAPI (blue). Representative fluorescence microscopy images of F-actin structures observed in ER-Hoxb8-derived OCs show examples of podosome cluster, podosome ring and podosome belt in cells differentiated on uncoated glass coverslips. A representative example of fluorescence microscopy images of mature F-actin ring structures in OCs which were differentiated on 100 mm dishes (300,000 cells) and subsequently plated on CaP-coated glass coverslips is shown in the lowest panel. Scale bars = 50 μm.
Mentions: In order to clarify whether ER-Hoxb8-derived OCs may be designated as a new source of bona fide OCs, we examined the functionality of these cells in comparison to OCs derived from conventional OC progenitor sources. Mature and functional OCs are known to develop an F-actin ring, which is important for the formation of a sealing zone that is associated with OC bone resorption activity [11]. Prior to the arrangement of a mature F-actin ring, actin structures of OCs pass through different intermediate developmental stages called podosome cluster, podosome ring and podosome belt [11,37]. To investigate these structures in ER-Hoxb8-derived OCs, cells were subjected to FITC-phalloidin staining at d5 of OC differentiation. Fig 5 illustrates podosome cluster, podosome ring and podosome belt structures in multi-nucleated cells (C3H/HeJ WT) differentiated on glass coverslips. These structures, which were also visible in BALB/c WT and p62 KO ER-Hoxb8-derived OCs (data now shown), were detectable in comparable quantities and qualities when cells were first differentiated and subsequently plated on biomimetic CaP-coated coverslips (data not shown). It is known that F-actin ring structures, which indicate actively resorbing OCs, are optimally visible on dentin or biomimetic bone substrates, for example on CaP-coated cell culture plates or glass coverslips, and thus fully developed F-actin rings are not likely to be detectable on non-coated plates or coverslips. In order to overcome these limitations, mature OCs, initially differentiated from ER-Hoxb8 progenitors in standard cell culture dishes, were isolated and subsequently plated on CaP-coated glass coverslips. Following incubation for 48 h, cells were stained for F-actin with FITC-phalloidin. Fig 5 (lowest panel) shows a representative multi-nucleated OC containing a heavily stained F-actin ring structure on the cell periphery. Cell movement and signs of OC-associated bone resorption activity at the interface of the F-actin ring are depicted in S1 Fig. The overview of merged bright-field, FICT-phalloidin and DAPI images from ER-Hoxb8-derived OCs (C3H/HeJ WT, p62 KO) points to a directional CaP resorption activity, which is clearly linked to and dependent on its F-actin ring formation on the cell periphery.

Bottom Line: 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.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.These cells represent a standardized and theoretically unlimited source for osteoclast-associated research projects.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus