Limits...
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

Effect of inhibitory and stimulatory cytokines on OC differentiation of primary BMs or ER-Hoxb8 SCs.(A) BALB/c BMMs or ER-Hoxb8 OC precursor cells of BALB/c and IL-4R KO origin (23,500 cells per cm2) were cultured with 1) M-CSF alone (“MФ”), 2) M-CSF and sRANKL (“OC”), or 3) the additional supplementation of indicated cytokines (GM-CSF: 10 ng/ml, remaining cytokines: 20 ng/ml). TRAP activity of supernatants was measured and compared to control differentiation (“OC”). Data are presented as the mean ± SD, n = 4. Significant differences of average TRAP activity compared to respective control values (“OC”) are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t-test). (B) Representative microscopy images illustrating morphology and TRAP staining of formalin-fixed cells after treatment with inhibitory and stimulatory modulators during OC differentiation. M-CSF-treated BMMs and Hoxb8 SCs as well as IL-4-treated BALB/c ER-Hoxb8 and BMMs do not show signs of TRAP staining. IL-4R KO cells are not sensitive to IL-4 and thus show normal OC differentiation potential. Scale bars = 150 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4631598&req=5

pone.0142211.g004: Effect of inhibitory and stimulatory cytokines on OC differentiation of primary BMs or ER-Hoxb8 SCs.(A) BALB/c BMMs or ER-Hoxb8 OC precursor cells of BALB/c and IL-4R KO origin (23,500 cells per cm2) were cultured with 1) M-CSF alone (“MФ”), 2) M-CSF and sRANKL (“OC”), or 3) the additional supplementation of indicated cytokines (GM-CSF: 10 ng/ml, remaining cytokines: 20 ng/ml). TRAP activity of supernatants was measured and compared to control differentiation (“OC”). Data are presented as the mean ± SD, n = 4. Significant differences of average TRAP activity compared to respective control values (“OC”) are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t-test). (B) Representative microscopy images illustrating morphology and TRAP staining of formalin-fixed cells after treatment with inhibitory and stimulatory modulators during OC differentiation. M-CSF-treated BMMs and Hoxb8 SCs as well as IL-4-treated BALB/c ER-Hoxb8 and BMMs do not show signs of TRAP staining. IL-4R KO cells are not sensitive to IL-4 and thus show normal OC differentiation potential. Scale bars = 150 μm.

Mentions: It is well known that OCs, which share similarities with macrophage-like foreign-body giant cells [35], and could be considered to be specialized giant macrophages [12], are sensitive to factors released from immune cells and directly interact with cells of the immune system [12]. To investigate the responsiveness of ER-Hoxb8-derived OCs to different OC-inhibitory cytokines (GM-CSF, IL-4, INF-γ) as wells as OC-stimulatory cytokines (IL-6, IL-15, TNF-α), both were tested during OC differentiation. Fig 4 illustrates TRAP activity of supernatants (Fig 4A) and cells (Fig 4B) derived from either primary BMMs (BALB/c WT) or BALB/c WT and IL-4R KO ER-Hoxb8 SCs at d4 (primary cells) or d5 (ER-Hoxb8 cells) of OC or macrophage (M-CSF) differentiation. As expected, M-CSF-treated cells (“MФ”) showed no detectable TRAP activity (Fig 4A and 4B), while M-CSF in combination with sRANKL (“OC”) resulted in high TRAP activity (Fig 4A and 4B). The addition of the inhibitory cytokine IL-4 (20 ng/ml) completely abolished OC differentiation and therefore TRAP secretion in primary BMM- as well as ER-Hoxb8-derived BALB/c WT cells (Fig 4A and 4B). This is consistent with published findings as, for example, Yamada and coworkers were able to show that IL-4 completely blocks osteoclastogenesis of mouse OC precursor cells [36]. Furthermore, we were successful in demonstrating the specificity of this IL-4 effect on ER-Hoxb8 cells since no inhibition of OC differentiation was detectable in IL-4R KO conditions (Fig 4A and 4B). The observed effects of the OC-inhibitory cytokines GM-CSF (10 ng/ml) and INF-γ (20 ng/ml) were comparable in the different cell lines used for OC differentiation (Fig 4A and 4B). TRAP-stained cells in Fig 4B reveal that stimulatory cytokines IL-6 and IL-15 similarly enhanced OC differentiation in all three OC precursor cell lines. However, we noticed that this effect was less pronounced based on the level of TRAP activity in the cell culture supernatants (Fig 4A). TNF-α administration enhanced OC differentiation of freshly isolated BMMs (Fig 4A and 4B), whereas unexpectedly this effect was not visible in TRAP-stained ER-Hoxb8-derived OCs. We further detected a markedly different level of TRAP activity in supernatants of TNF-α-treated IL-4R KO and BALB/c WT ER-Hoxb8-derived OC differentiation conditions. These unexpected differential effects of TNF-α treatment on ER-Hoxb8 cells are currently under in-depth investigation.


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

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

Effect of inhibitory and stimulatory cytokines on OC differentiation of primary BMs or ER-Hoxb8 SCs.(A) BALB/c BMMs or ER-Hoxb8 OC precursor cells of BALB/c and IL-4R KO origin (23,500 cells per cm2) were cultured with 1) M-CSF alone (“MФ”), 2) M-CSF and sRANKL (“OC”), or 3) the additional supplementation of indicated cytokines (GM-CSF: 10 ng/ml, remaining cytokines: 20 ng/ml). TRAP activity of supernatants was measured and compared to control differentiation (“OC”). Data are presented as the mean ± SD, n = 4. Significant differences of average TRAP activity compared to respective control values (“OC”) are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t-test). (B) Representative microscopy images illustrating morphology and TRAP staining of formalin-fixed cells after treatment with inhibitory and stimulatory modulators during OC differentiation. M-CSF-treated BMMs and Hoxb8 SCs as well as IL-4-treated BALB/c ER-Hoxb8 and BMMs do not show signs of TRAP staining. IL-4R KO cells are not sensitive to IL-4 and thus show normal OC differentiation potential. Scale bars = 150 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142211.g004: Effect of inhibitory and stimulatory cytokines on OC differentiation of primary BMs or ER-Hoxb8 SCs.(A) BALB/c BMMs or ER-Hoxb8 OC precursor cells of BALB/c and IL-4R KO origin (23,500 cells per cm2) were cultured with 1) M-CSF alone (“MФ”), 2) M-CSF and sRANKL (“OC”), or 3) the additional supplementation of indicated cytokines (GM-CSF: 10 ng/ml, remaining cytokines: 20 ng/ml). TRAP activity of supernatants was measured and compared to control differentiation (“OC”). Data are presented as the mean ± SD, n = 4. Significant differences of average TRAP activity compared to respective control values (“OC”) are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t-test). (B) Representative microscopy images illustrating morphology and TRAP staining of formalin-fixed cells after treatment with inhibitory and stimulatory modulators during OC differentiation. M-CSF-treated BMMs and Hoxb8 SCs as well as IL-4-treated BALB/c ER-Hoxb8 and BMMs do not show signs of TRAP staining. IL-4R KO cells are not sensitive to IL-4 and thus show normal OC differentiation potential. Scale bars = 150 μm.
Mentions: It is well known that OCs, which share similarities with macrophage-like foreign-body giant cells [35], and could be considered to be specialized giant macrophages [12], are sensitive to factors released from immune cells and directly interact with cells of the immune system [12]. To investigate the responsiveness of ER-Hoxb8-derived OCs to different OC-inhibitory cytokines (GM-CSF, IL-4, INF-γ) as wells as OC-stimulatory cytokines (IL-6, IL-15, TNF-α), both were tested during OC differentiation. Fig 4 illustrates TRAP activity of supernatants (Fig 4A) and cells (Fig 4B) derived from either primary BMMs (BALB/c WT) or BALB/c WT and IL-4R KO ER-Hoxb8 SCs at d4 (primary cells) or d5 (ER-Hoxb8 cells) of OC or macrophage (M-CSF) differentiation. As expected, M-CSF-treated cells (“MФ”) showed no detectable TRAP activity (Fig 4A and 4B), while M-CSF in combination with sRANKL (“OC”) resulted in high TRAP activity (Fig 4A and 4B). The addition of the inhibitory cytokine IL-4 (20 ng/ml) completely abolished OC differentiation and therefore TRAP secretion in primary BMM- as well as ER-Hoxb8-derived BALB/c WT cells (Fig 4A and 4B). This is consistent with published findings as, for example, Yamada and coworkers were able to show that IL-4 completely blocks osteoclastogenesis of mouse OC precursor cells [36]. Furthermore, we were successful in demonstrating the specificity of this IL-4 effect on ER-Hoxb8 cells since no inhibition of OC differentiation was detectable in IL-4R KO conditions (Fig 4A and 4B). The observed effects of the OC-inhibitory cytokines GM-CSF (10 ng/ml) and INF-γ (20 ng/ml) were comparable in the different cell lines used for OC differentiation (Fig 4A and 4B). TRAP-stained cells in Fig 4B reveal that stimulatory cytokines IL-6 and IL-15 similarly enhanced OC differentiation in all three OC precursor cell lines. However, we noticed that this effect was less pronounced based on the level of TRAP activity in the cell culture supernatants (Fig 4A). TNF-α administration enhanced OC differentiation of freshly isolated BMMs (Fig 4A and 4B), whereas unexpectedly this effect was not visible in TRAP-stained ER-Hoxb8-derived OCs. We further detected a markedly different level of TRAP activity in supernatants of TNF-α-treated IL-4R KO and BALB/c WT ER-Hoxb8-derived OC differentiation conditions. These unexpected differential effects of TNF-α treatment on ER-Hoxb8 cells are currently under in-depth investigation.

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