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Shuo Chen - One of the best experts on this subject based on the ideXlab platform.
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establishment of an immortalized mouse bmp2 knockout Dental Papilla mesenchymal cell line
Methods of Molecular Biology, 2019Co-Authors: Shuo ChenAbstract:Bone morphogenetic protein 2 (Bmp2) is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta (DGI), showing Dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain primary Bmp2 cKO Dental Papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 Dental Papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. Here we describe the generation of an immortalized deleted Bmp2 Dental Papilla mesenchymal (iBmp2ko/ko-dp) cell line by introducing Cre fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 Dental Papilla mesenchymal (iBmp2flox/flox-dp) cells.
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klf10 regulates odontoblast differentiation and mineralization via promoting expression of dentin matrix protein 1 and dentin sialophosphoprotein genes
Cell and Tissue Research, 2016Co-Authors: Zhuo Chen, Wentong Li, Han Wang, Shuli Deng, Hui Chen, Shuo ChenAbstract:Klf10, a member of the Kruppel-like family of transcription factors, is critical for osteoblast differentiation, bone formation and mineralization. However, whether Klf10 is involved in odontoblastic differentiation and tooth development has not been determined. In this study, we investigate the expression patterns of Klf10 during murine tooth development in vivo and its role in odontoblastic differentiation in vitro. Klf10 protein was expressed in the enamel organ and the underlying mesenchyme, ameloblasts and odontoblasts at early and later stages of murine molar formation. Furthermore, the expression of Klf10, Dmp1, Dspp and Runx2 was significantly elevated during the process of mouse Dental Papilla mesenchymal differentiation and mineralization. The overexpression of Klf10 induced Dental Papilla mesenchymal cell differentiation and mineralization as detected by alkaline phosphatase staining and alizarin red S assay. Klf10 additionally up-regulated the expression of odontoblastic differentiation marker genes Dmp1, Dspp and Runx2 in mouse Dental Papilla mesenchymal cells. The molecular mechanism of Klf10 in controlling Dmp1 and Dspp expression is thus to activate their regulatory regions in a dosage-dependent manner. Our results suggest that Klf10 is involved in tooth development and promotes odontoblastic differentiation via the up-regulation of Dmp1 and Dspp transcription.
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immortalized mouse floxed fam20c Dental Papillar mesenchymal and osteoblast cell lines retain their primary characteristics
Journal of Cellular Physiology, 2015Co-Authors: Xiaofang Wang, Priyam Jani, Yongbo Lu, Hua Zhang, Shuo ChenAbstract:Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c Dental Papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c Dental Papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T-antigen (SV40 T-Ag). The immortalization of floxed Fam20c Dental Papilla mesenchymal cells and osteoblasts was verified by the long-term passages and genomic integration of SV40 T-Ag. The immortalized floxed Fam20c Dental Papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary Dental Papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c Dental Papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized Dental Papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c Dental Papilla mesenchymal and osteoblast cell lines. J. Cell. Physiol. 9999: 2581–2587, 2015. © 2015 Wiley Periodicals, Inc.
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establishment of immortalized mouse bmp2 knock out Dental Papilla mesenchymal cells necessary for study of odontoblastic differentiation and odontogenesis
Journal of Cellular Physiology, 2015Co-Authors: Kevin J Donly, S E Harris, Feng Wang, Chunyan Wan, Daoshu Luo, Mary Macdougall, Shuo ChenAbstract:Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing Dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO Dental Papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 Dental Papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 Dental Papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 Dental Papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating Dental Papilla mesenchymal cell lineages.
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immortalized mouse Dental Papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2
In Vitro Cellular & Developmental Biology – Animal, 2013Co-Authors: Feng Wang, Yuan Yang, Feng Guo, Qingping Gao, Huihsiu Chuang, Lisa Shoff, Weiwei Wang, Shuo ChenAbstract:Odontogenesis is the result of the reciprocal interactions between epithelial–mesenchymal cells leading to terminally differentiated odontoblasts. This process from Dental Papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the Dental mesenchymal cell differentiation into odontoblasts and the molecular events of dentin formation. In this study, immortalized Dental Papilla mesenchymal cell lines were generated from the first mouse mandibular molars at postnatal day 3 using pSV40. These transformed cells were characterized by RT-PCR, immunohistochemistry, Western blot, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iMDP-3, displayed a high proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralized nodules. Furthermore, iMDP-3 cells had high transfection efficiency as well as were inducible and responded to BMP2 stimulation. We conclude that the establishment of the stable murine Dental Papilla mesenchymal cell line might be used for studying the mechanisms of Dental cell differentiation and dentin formation.
Junsheng Feng - One of the best experts on this subject based on the ideXlab platform.
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immortalized mouse floxed bmp2 Dental Papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to bmp2
Journal of Cellular Physiology, 2010Co-Authors: Li An Wu, Junsheng Feng, Lynn Wang, Yan Dong Mu, Andrew Baker, Kevin J Donly, Jelica Gluhakheinrich, S E HarrisAbstract:Tooth development involves sequential and reciprocal interactions between Dental epithelial and mesenchymal cells, and proceeds through a series of cytodifferentiations in specific spatial-temporal patterns (Linde and Goldberg, 1993). Dentinogenesis is a complex process in which multiple signaling pathways converge to induce dentin formation and is controlled by many growth and transcription factors (Thesleff, 2003). The bone morphogenetic proteins (Bmps) are structurally related to the transforming growth factor beta (TGF-β) superfamily and were originally identified by their capacity to induce ectopic bone formation in rodents (Urist, 1965; Wozney et al., 1988). Members of the Bmp family have diverse biological functions during embryonic development (Hogan, 1996; Wu et al., 2003) including a vital role in osteogenesis (Chen et al., 2004; Rosen, 2009). Among the Bmp family members, Bmp2 has been extensively studied for its various biological functions during chondrogenic and osteogenic differentiation (Reddi, 1997; Ducy and Karsenty, 2000). Also, Bmp2 has been shown to promote Dental pulp stem cell commitment to the odontoblast lineage in vitro (Yang et al., 2009) and induces Dental pulp cell differentiation and mineralization in vitro and in vivo (Nakashima, 2005; Chen et al., 2008). However, detail understandings of the molecular mechanisms of Bmp2 exerting its effects on tooth development and formation remain elusive in particular during postnatal tooth development as homozygous mutant embryos for Bmp2 show developmental abnormalities and die at embryo day 9.5 (Zhang and Bradley, 1996). Recently, conditional Bmp2 knock out (cBmp2-KO) mice were generated and revealed important roles of Bmp2 in later stages of osteogenesis (Bandyopadhyay et al., 2006) and bone fracture healing (Tsuji et al., 2006) as well as other organ development (Ma et al., 2005; Rivera-Feliciano and Tabin, 2006; Lee et al., 2007; Singh et al., 2008). However, roles of Bmp2 during tooth development and formation have not been completely understood. Unlike bone and other tissues, it is relatively hard to collect enough amounts of Dental tissues from a single tooth. Therefore, generation of a floxed Bmp2 Dental Papilla mesenchymal cell line would be a valuable tool for studying the effects of Bmp2 on Dental cell lineages as well as relevant molecular events involved in matrix mineralization and dentin regeneration. Such information will help realize the potential of BMP2 as therapeutic agent and for the rational targeting of specific Bmp2 to the appropriate clinical indication. In this study, we established an immortalized mouse floxed Bmp2 Dental Papilla mesenchymal cell line using transduction of simian phenotypic and virus 40 T-antigen (SV40). We further observed these cell growth rates and their genotypic and phenotypic characteristics as compared to primary cells. Finally, we tested whether these immortalized cells were inducible by growth factors.
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immortalized mouse floxed bmp2 Dental Papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to bmp2
Journal of Cellular Physiology, 2010Co-Authors: Junsheng Feng, Lynn Wang, Andrew Baker, Kevin J Donly, Jelica Gluhakheinrich, S E Harris, Mary Macdougall, Shuo ChenAbstract:Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 Dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on Dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of Dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of Dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 Dental Papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT-PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2-dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2-dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 Dental Papilla mesenchyma cell line that might be used for studying the mechanisms of Dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways.
S E Harris - One of the best experts on this subject based on the ideXlab platform.
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establishment of immortalized mouse bmp2 knock out Dental Papilla mesenchymal cells necessary for study of odontoblastic differentiation and odontogenesis
Journal of Cellular Physiology, 2015Co-Authors: Kevin J Donly, S E Harris, Feng Wang, Chunyan Wan, Daoshu Luo, Mary Macdougall, Shuo ChenAbstract:Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing Dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO Dental Papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 Dental Papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 Dental Papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 Dental Papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating Dental Papilla mesenchymal cell lineages.
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immortalized mouse floxed bmp2 Dental Papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to bmp2
Journal of Cellular Physiology, 2010Co-Authors: Li An Wu, Junsheng Feng, Lynn Wang, Yan Dong Mu, Andrew Baker, Kevin J Donly, Jelica Gluhakheinrich, S E HarrisAbstract:Tooth development involves sequential and reciprocal interactions between Dental epithelial and mesenchymal cells, and proceeds through a series of cytodifferentiations in specific spatial-temporal patterns (Linde and Goldberg, 1993). Dentinogenesis is a complex process in which multiple signaling pathways converge to induce dentin formation and is controlled by many growth and transcription factors (Thesleff, 2003). The bone morphogenetic proteins (Bmps) are structurally related to the transforming growth factor beta (TGF-β) superfamily and were originally identified by their capacity to induce ectopic bone formation in rodents (Urist, 1965; Wozney et al., 1988). Members of the Bmp family have diverse biological functions during embryonic development (Hogan, 1996; Wu et al., 2003) including a vital role in osteogenesis (Chen et al., 2004; Rosen, 2009). Among the Bmp family members, Bmp2 has been extensively studied for its various biological functions during chondrogenic and osteogenic differentiation (Reddi, 1997; Ducy and Karsenty, 2000). Also, Bmp2 has been shown to promote Dental pulp stem cell commitment to the odontoblast lineage in vitro (Yang et al., 2009) and induces Dental pulp cell differentiation and mineralization in vitro and in vivo (Nakashima, 2005; Chen et al., 2008). However, detail understandings of the molecular mechanisms of Bmp2 exerting its effects on tooth development and formation remain elusive in particular during postnatal tooth development as homozygous mutant embryos for Bmp2 show developmental abnormalities and die at embryo day 9.5 (Zhang and Bradley, 1996). Recently, conditional Bmp2 knock out (cBmp2-KO) mice were generated and revealed important roles of Bmp2 in later stages of osteogenesis (Bandyopadhyay et al., 2006) and bone fracture healing (Tsuji et al., 2006) as well as other organ development (Ma et al., 2005; Rivera-Feliciano and Tabin, 2006; Lee et al., 2007; Singh et al., 2008). However, roles of Bmp2 during tooth development and formation have not been completely understood. Unlike bone and other tissues, it is relatively hard to collect enough amounts of Dental tissues from a single tooth. Therefore, generation of a floxed Bmp2 Dental Papilla mesenchymal cell line would be a valuable tool for studying the effects of Bmp2 on Dental cell lineages as well as relevant molecular events involved in matrix mineralization and dentin regeneration. Such information will help realize the potential of BMP2 as therapeutic agent and for the rational targeting of specific Bmp2 to the appropriate clinical indication. In this study, we established an immortalized mouse floxed Bmp2 Dental Papilla mesenchymal cell line using transduction of simian phenotypic and virus 40 T-antigen (SV40). We further observed these cell growth rates and their genotypic and phenotypic characteristics as compared to primary cells. Finally, we tested whether these immortalized cells were inducible by growth factors.
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immortalized mouse floxed bmp2 Dental Papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to bmp2
Journal of Cellular Physiology, 2010Co-Authors: Junsheng Feng, Lynn Wang, Andrew Baker, Kevin J Donly, Jelica Gluhakheinrich, S E Harris, Mary Macdougall, Shuo ChenAbstract:Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 Dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on Dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of Dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of Dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 Dental Papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT-PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2-dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2-dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 Dental Papilla mesenchyma cell line that might be used for studying the mechanisms of Dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways.
Kevin J Donly - One of the best experts on this subject based on the ideXlab platform.
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establishment of immortalized mouse bmp2 knock out Dental Papilla mesenchymal cells necessary for study of odontoblastic differentiation and odontogenesis
Journal of Cellular Physiology, 2015Co-Authors: Kevin J Donly, S E Harris, Feng Wang, Chunyan Wan, Daoshu Luo, Mary Macdougall, Shuo ChenAbstract:Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing Dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO Dental Papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 Dental Papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 Dental Papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 Dental Papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating Dental Papilla mesenchymal cell lineages.
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immortalized mouse floxed bmp2 Dental Papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to bmp2
Journal of Cellular Physiology, 2010Co-Authors: Li An Wu, Junsheng Feng, Lynn Wang, Yan Dong Mu, Andrew Baker, Kevin J Donly, Jelica Gluhakheinrich, S E HarrisAbstract:Tooth development involves sequential and reciprocal interactions between Dental epithelial and mesenchymal cells, and proceeds through a series of cytodifferentiations in specific spatial-temporal patterns (Linde and Goldberg, 1993). Dentinogenesis is a complex process in which multiple signaling pathways converge to induce dentin formation and is controlled by many growth and transcription factors (Thesleff, 2003). The bone morphogenetic proteins (Bmps) are structurally related to the transforming growth factor beta (TGF-β) superfamily and were originally identified by their capacity to induce ectopic bone formation in rodents (Urist, 1965; Wozney et al., 1988). Members of the Bmp family have diverse biological functions during embryonic development (Hogan, 1996; Wu et al., 2003) including a vital role in osteogenesis (Chen et al., 2004; Rosen, 2009). Among the Bmp family members, Bmp2 has been extensively studied for its various biological functions during chondrogenic and osteogenic differentiation (Reddi, 1997; Ducy and Karsenty, 2000). Also, Bmp2 has been shown to promote Dental pulp stem cell commitment to the odontoblast lineage in vitro (Yang et al., 2009) and induces Dental pulp cell differentiation and mineralization in vitro and in vivo (Nakashima, 2005; Chen et al., 2008). However, detail understandings of the molecular mechanisms of Bmp2 exerting its effects on tooth development and formation remain elusive in particular during postnatal tooth development as homozygous mutant embryos for Bmp2 show developmental abnormalities and die at embryo day 9.5 (Zhang and Bradley, 1996). Recently, conditional Bmp2 knock out (cBmp2-KO) mice were generated and revealed important roles of Bmp2 in later stages of osteogenesis (Bandyopadhyay et al., 2006) and bone fracture healing (Tsuji et al., 2006) as well as other organ development (Ma et al., 2005; Rivera-Feliciano and Tabin, 2006; Lee et al., 2007; Singh et al., 2008). However, roles of Bmp2 during tooth development and formation have not been completely understood. Unlike bone and other tissues, it is relatively hard to collect enough amounts of Dental tissues from a single tooth. Therefore, generation of a floxed Bmp2 Dental Papilla mesenchymal cell line would be a valuable tool for studying the effects of Bmp2 on Dental cell lineages as well as relevant molecular events involved in matrix mineralization and dentin regeneration. Such information will help realize the potential of BMP2 as therapeutic agent and for the rational targeting of specific Bmp2 to the appropriate clinical indication. In this study, we established an immortalized mouse floxed Bmp2 Dental Papilla mesenchymal cell line using transduction of simian phenotypic and virus 40 T-antigen (SV40). We further observed these cell growth rates and their genotypic and phenotypic characteristics as compared to primary cells. Finally, we tested whether these immortalized cells were inducible by growth factors.
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immortalized mouse floxed bmp2 Dental Papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to bmp2
Journal of Cellular Physiology, 2010Co-Authors: Junsheng Feng, Lynn Wang, Andrew Baker, Kevin J Donly, Jelica Gluhakheinrich, S E Harris, Mary Macdougall, Shuo ChenAbstract:Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 Dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on Dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of Dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of Dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 Dental Papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT-PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2-dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2-dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 Dental Papilla mesenchyma cell line that might be used for studying the mechanisms of Dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways.
Li An Wu - One of the best experts on this subject based on the ideXlab platform.
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immortalized mouse floxed bmp2 Dental Papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to bmp2
Journal of Cellular Physiology, 2010Co-Authors: Li An Wu, Junsheng Feng, Lynn Wang, Yan Dong Mu, Andrew Baker, Kevin J Donly, Jelica Gluhakheinrich, S E HarrisAbstract:Tooth development involves sequential and reciprocal interactions between Dental epithelial and mesenchymal cells, and proceeds through a series of cytodifferentiations in specific spatial-temporal patterns (Linde and Goldberg, 1993). Dentinogenesis is a complex process in which multiple signaling pathways converge to induce dentin formation and is controlled by many growth and transcription factors (Thesleff, 2003). The bone morphogenetic proteins (Bmps) are structurally related to the transforming growth factor beta (TGF-β) superfamily and were originally identified by their capacity to induce ectopic bone formation in rodents (Urist, 1965; Wozney et al., 1988). Members of the Bmp family have diverse biological functions during embryonic development (Hogan, 1996; Wu et al., 2003) including a vital role in osteogenesis (Chen et al., 2004; Rosen, 2009). Among the Bmp family members, Bmp2 has been extensively studied for its various biological functions during chondrogenic and osteogenic differentiation (Reddi, 1997; Ducy and Karsenty, 2000). Also, Bmp2 has been shown to promote Dental pulp stem cell commitment to the odontoblast lineage in vitro (Yang et al., 2009) and induces Dental pulp cell differentiation and mineralization in vitro and in vivo (Nakashima, 2005; Chen et al., 2008). However, detail understandings of the molecular mechanisms of Bmp2 exerting its effects on tooth development and formation remain elusive in particular during postnatal tooth development as homozygous mutant embryos for Bmp2 show developmental abnormalities and die at embryo day 9.5 (Zhang and Bradley, 1996). Recently, conditional Bmp2 knock out (cBmp2-KO) mice were generated and revealed important roles of Bmp2 in later stages of osteogenesis (Bandyopadhyay et al., 2006) and bone fracture healing (Tsuji et al., 2006) as well as other organ development (Ma et al., 2005; Rivera-Feliciano and Tabin, 2006; Lee et al., 2007; Singh et al., 2008). However, roles of Bmp2 during tooth development and formation have not been completely understood. Unlike bone and other tissues, it is relatively hard to collect enough amounts of Dental tissues from a single tooth. Therefore, generation of a floxed Bmp2 Dental Papilla mesenchymal cell line would be a valuable tool for studying the effects of Bmp2 on Dental cell lineages as well as relevant molecular events involved in matrix mineralization and dentin regeneration. Such information will help realize the potential of BMP2 as therapeutic agent and for the rational targeting of specific Bmp2 to the appropriate clinical indication. In this study, we established an immortalized mouse floxed Bmp2 Dental Papilla mesenchymal cell line using transduction of simian phenotypic and virus 40 T-antigen (SV40). We further observed these cell growth rates and their genotypic and phenotypic characteristics as compared to primary cells. Finally, we tested whether these immortalized cells were inducible by growth factors.