A periosteum-derived cell line to study the role of BMP/TGFβ signaling in periosteal cell behavior and function

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors

The Role of IGFBP4 in Regulating Bone and Adipose Tissue Development in Mice

Insulin-like Growth Factor (IGF) regulates bone growth and induces adipogenesis. Interestingly, IGF Binding Protein 4 (IGFBP4) is highly expressed in adipocytes and osteoblasts and was found to be inhibitory of IGF in vitro. However, studies in mice suggested that it does not act as an inhibitor in vivo. The purpose of our study is to clarify how IGFBP4 mediates adipose and skeletal development in vivo in Igfbp4 null (Igfbp4-/-) mice. Adult Igfbp4-/- mice suffered from growth retardation with reductions in body weight, body length and femur length. At 8 and 16 weeks of age, DXA showed that both sexes of Igfbp4-/- mice had reduced fat proportion and lean mass. The weights of inguinal and gonadal adipose depots were significantly reduced. Pparγ expression was significantly decreased in inguinal fat from Igfbp4-/- male and female mice indicating that lipid metabolism may be altered with the deletion of IGFBP4. Indeed, cultures of ear mesenchymal stem cells (eMSC) and bone marrow stromal cells (BMSC) from Igfbp4-/- mice showed decreased adipogenesis. Moreover, control eMSC increased their levels of phosphorylated Akt (p-Akt) and Igfbp4 expression during adipogenesis. However, Igfbp4-/- eMSC had decreased levels of p-Akt suggesting that IGF signaling may be impaired with loss of Igfbp4. When mice were challenged with a high fat diet (HFD), Igfbp4-/- females were protected against HFD-induced obesity. However, Igfbp4-/- males gained as much weight than littermate controls marked with adipocyte hypertrophy, liver steatosis and brown fat lipid accumulation. After high fat feeding, inguinal Igfbp4 expression of control mice was downregulated in females but not in males suggesting a clear sex-specific regulation of Igfbp4. At 16 weeks of age and on a regular diet, marked reductions in bone mineral density and bone mineral content were observed in Igfbp4-/- females, but not in males. Micro-computed Tomography (μCT) of femurs revealed reductions in trabecular bone volume and trabecular and cortical thicknesses in Igfbp4-/- females. Surprisingly, males had significantly more trabecular bone with higher connectivity density and trabecular number. Concordantly, Sost, an inhibitor of bone formation, had decreased gene expression in Igfbp4-/- males but not in females. In summary, our results indicate that loss of Igfbp4 impairs mesenchymal differentiation and IGF downstream signaling. Our data shows that IGFBP4 modulates bone and fat development with a clear gender and tissue specificity

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays

IGFBP4 is required for adipogenesis and influences the distribution of adipose depots.

Insulinlike growth factor (IGF) I induces adipogenesis in vitro. IGF-binding protein 4 (IGFBP4) is highly expressed in adipocytes and osteoblasts and is inhibitory of IGFs in vitro. We previously reported that Igfbp4 null mice (Igfbp4-/-) had decreased fat proportions at 8 and 16 weeks of age. However, the mechanism leading to the reduced adiposity remains unknown. The purpose of this study was to elucidate how IGFBP4 mediates adipose tissue development in vivo. Our results showed that inguinal and gonadal white adipose tissue (gWAT) from Igfbp4-/- mice had decreased weights and Pparγ expression. Cultures of primary bone marrow stromal cells (BMSCs) and ear mesenchymal stem cells (eMSCs) from mutant mice showed reduced adipogenesis. Both BMSCs and eMSC had a strong induction of Igfbp4 expression during adipogenesis. Furthermore, the increase in phosphorylated Akt (p-Akt), a downstream target of IGF-I signaling, in wild-type cells, was blunted in mutant eMSCs. On a high-fat diet (HFD) there were sexual differences in adipocyte expansion of Igfbp4-/- mice. Mutant males gained weight by expanding their white fat depots. However, Igfbp4-/- female mice were protected against diet-induced obesity. Ovariectomized Igfbp4-/- female mice gained weight in a manner similar to that seen in ovariectomized controls. Thus, Igfbp4 is required for inguinal fat expansion in female mice but not in male mice. However, gWAT expansion, which is prevented by estrogen during HFD, does not require Igfbp4

Image3_A periosteum-derived cell line to study the role of BMP/TGFβ signaling in periosteal cell behavior and function.TIF

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors.</p

IGFBP-4 regulates adult skeletal growth in a sex-specific manner.

Insulin-like growth factor-1 (IGF-1) and its binding proteins are critical mediators of skeletal growth. Insulin-like growth factor-binding protein 4 (IGFBP-4) is highly expressed in osteoblasts and inhibits IGF-1 action

Image4_A periosteum-derived cell line to study the role of BMP/TGFβ signaling in periosteal cell behavior and function.TIF

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors.</p

Image1_A periosteum-derived cell line to study the role of BMP/TGFβ signaling in periosteal cell behavior and function.tif

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors.</p

Igfbp2 deletion in ovariectomized mice enhances energy expenditure but accelerates bone loss.

Previously, we reported sexually dimorphic bone mass and body composition phenotypes in Igfbp2(-/-) mice (-/-), where male mice exhibited decreased bone and increased fat mass, whereas female mice displayed increased bone but no changes in fat mass. To investigate the interaction between IGF-binding protein (IGFBP)-2 and estrogen, we subjected Igfbp2 -/- and +/+ female mice to ovariectomy (OVX) or sham surgery at 8 weeks of age. At 20 weeks of age, mice underwent metabolic cage analysis and insulin tolerance tests before killing. At harvest, femurs were collected for microcomputed tomography, serum for protein levels, brown adipose tissue (BAT) and inguinal white adipose tissue (IWAT) adipose depots for histology, gene expression, and mitochondrial respiration analysis of whole tissue. In +/+ mice, serum IGFBP-2 dropped 30% with OVX. In the absence of IGFBP-2, OVX had no effect on preformed BAT; however, there was significant browning of the IWAT depot coinciding with less weight gain, increased insulin sensitivity, lower intraabdominal fat, and increased bone loss due to higher resorption and lower formation. Likewise, after OVX, energy expenditure, physical activity and BAT mitochondrial respiration were decreased less in the OVX-/- compared with OVX+/+. Mitochondrial respiration of IWAT was reduced in OVX+/+ yet remained unchanged in OVX-/- mice. These changes were associated with significant increases in Fgf21 and Foxc2 expression, 2 proteins known for their insulin sensitizing and browning of WAT effects. We conclude that estrogen deficiency has a profound effect on body and bone composition in the absence of IGFBP-2 and may be related to changes in fibroblast growth factor 21

Image6_A periosteum-derived cell line to study the role of BMP/TGFβ signaling in periosteal cell behavior and function.TIF

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors.</p