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1.
J Periodontal Res ; 57(1): 162-172, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34826339

ABSTRACT

BACKGROUND AND OBJECTIVE: Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. METHODS: Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (µCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. RESULTS: The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. CONCLUSIONS: HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.


Subject(s)
Alveolar Bone Loss , Exosomes , Periodontitis , Alveolar Bone Loss/diagnostic imaging , Alveolar Bone Loss/therapy , Animals , Cell Differentiation , Dental Pulp , Mice , Periodontitis/therapy , X-Ray Microtomography
2.
J Bone Miner Metab ; 37(3): 467-474, 2019 May.
Article in English | MEDLINE | ID: mdl-30187276

ABSTRACT

The central nervous system in adult mammals does not heal spontaneously after spinal cord injury (SCI). However, SCI treatment has been improved recently following the development of cell transplantation therapy. We recently reported that fibroblast growth factor (FGF) 2-pretreated human dental pulp cells (hDPCs) can improve recovery in a rat model of SCI. This study aimed to investigate mechanisms underlying the curative effect of SCI enhanced via FGF2 pretreatment; we selected three hDPC lines upon screening for the presence of mesenchymal stem cell markers and of their functionality in a rat model of SCI, as assessed using the Basso, Beattie, and Bresnahan score of locomotor functional scale, electrophysiological tests, and morphological analyses. We identified FGF2-responsive genes via gene expression analyses in these lines. FGF2 treatment upregulated GABRB1, MMP1, and DRD2, which suggested to contribute to SCI or central the nervous system. In an expanded screening of additional lines, GABRB1 displayed rather unique and interesting behavior; two lines with the lowest sensitivity of GABRB1 to FGF2 treatment displayed an extremely minor effect in the SCI model. These findings provide insights into the role of FGF2-responsive genes, especially GABRB1, in recovery from SCI, using hDPCs treated with FGF2.


Subject(s)
Dental Pulp/cytology , Fibroblast Growth Factor 2/pharmacology , Gene Expression Regulation/drug effects , Spinal Cord Injuries/genetics , Spinal Cord Injuries/therapy , Animals , Disease Models, Animal , Electrophysiological Phenomena/drug effects , Humans , Motor Activity/drug effects , Rats , Rats, Sprague-Dawley , Recovery of Function , Spinal Cord Injuries/physiopathology
3.
Sci Rep ; 7(1): 13500, 2017 10 18.
Article in English | MEDLINE | ID: mdl-29044129

ABSTRACT

Human dental pulp cells (DPCs), adherent cells derived from dental pulp tissues, are potential tools for cell transplantation therapy. However, little work has been done to optimize such transplantation. In this study, DPCs were treated with fibroblast growth factor-2 (FGF2) for 5-6 consecutive serial passages and were transplanted into the injury site immediately after complete transection of the rat spinal cord. FGF2 priming facilitated the DPCs to promote axonal regeneration and to improve locomotor function in the rat with spinal cord injury (SCI). Additional analyses revealed that FGF2 priming protected cultured DPCs from hydrogen-peroxide-induced cell death and increased the number of DPCs in the SCI rat spinal cord even 7 weeks after transplantation. The production of major neurotrophic factors was equivalent in FGF2-treated and untreated DPCs. These observations suggest that FGF2 priming might protect DPCs from the post-trauma microenvironment in which DPCs infiltrate and resident immune cells generate cytotoxic reactive oxygen species. Surviving DPCs could increase the availability of neurotrophic factors in the lesion site, thereby promoting axonal regeneration and locomotor function recovery.


Subject(s)
Dental Pulp/cytology , Fibroblast Growth Factor 2/pharmacology , Mesenchymal Stem Cell Transplantation/methods , Nerve Regeneration , Spinal Cord Injuries/therapy , Animals , Axon Guidance , Cells, Cultured , Female , Humans , Locomotion , Mesenchymal Stem Cells/drug effects , Rats , Rats, Wistar
4.
Sci Rep ; 4: 7283, 2014 Dec 04.
Article in English | MEDLINE | ID: mdl-25471527

ABSTRACT

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by defined transcription factors has been a well-established technique and will provide an invaluable resource for regenerative medicine. However, the low reprogramming efficiency of human iPSC is still a limitation for clinical application. Here we showed that the reprogramming potential of human dental pulp cells (DPCs) obtained from immature teeth is much higher than those of mature teeth DPCs. Furthermore, immature teeth DPCs can be reprogrammed by OCT3/4 and SOX2, conversely these two factors are insufficient to convert mature teeth DPCs to pluripotent states. Using a gene expression profiles between these two DPC groups, we identified a new transcript factor, distal-less homeobox 4 (DLX4), which was highly expressed in immature teeth DPCs and significantly promoted human iPSC generation in combination with OCT3/4, SOX2, and KLF4. We further show that activation of TGF-ß signaling suppresses the expression of DLX4 in DPCs and impairs the iPSC generation of DPCs. Our findings indicate that DLX4 can functionally replace c-MYC and supports efficient reprogramming of immature teeth DPCs.


Subject(s)
Genes, Homeobox/genetics , Homeodomain Proteins/genetics , Induced Pluripotent Stem Cells/physiology , Transcription Factors/genetics , Cells, Cultured , Cellular Reprogramming/genetics , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Octamer Transcription Factor-3/genetics , Proto-Oncogene Proteins c-myc/genetics , SOXB1 Transcription Factors/genetics , Transcriptome/genetics , Transforming Growth Factor beta/genetics
5.
PLoS One ; 9(12): e115392, 2014.
Article in English | MEDLINE | ID: mdl-25521610

ABSTRACT

Human dental pulp cells (hDPCs) are a promising resource for regenerative medicine and tissue engineering and can be used for derivation of induced pluripotent stem cells (iPSCs). However, current protocols use reagents of animal origin (mainly fetal bovine serum, FBS) that carry the potential risk of infectious diseases and unwanted immunogenicity. Here, we report a chemically defined protocol to isolate and maintain the growth and differentiation potential of hDPCs. hDPCs cultured under these conditions showed significantly less primary colony formation than those with FBS. Cell culture under stringently defined conditions revealed a donor-dependent growth capacity; however, once established, the differentiation capabilities of the hDPCs were comparable to those observed with FBS. DNA array analyses indicated that the culture conditions robustly altered hDPC gene expression patterns but, more importantly, had little effect on neither pluripotent gene expression nor the efficiency of iPSC induction. The chemically defined culture conditions described herein are not perfect serum replacements, but can be used for the safe establishment of iPSCs and will find utility in applications for cell-based regenerative medicine.


Subject(s)
Dental Pulp/cytology , Induced Pluripotent Stem Cells/cytology , Adolescent , Cell Differentiation , Cells, Cultured , Humans , Primary Cell Culture/methods
6.
Dev Growth Differ ; 55(2): 270-81, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23347447

ABSTRACT

Melanocytes are pigment-producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. The widely accepted premise that NCCs migrating along the dorsolateral pathway are the main source of melanocytes in the skin was recently challenged by the finding that Schwann cell precursors are the major cellular source of melanocytes in the skin. Still, in a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In this study, we show that a NCC population that is not derived from Sox1(+) dorsal neuroepithelial cells but are derived from Sox1(-) cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1(-) cells clearly segregate from cells that originated from Sox1(+) dorsal neuroepithelial cell-derived NCCs. The possible derivation of Sox1(-) cells from epidermal cells also strengthens their non-neuroepithelial origin.


Subject(s)
Melanocytes/metabolism , Neural Crest/metabolism , SOXB1 Transcription Factors/metabolism , Schwann Cells/metabolism , Skin/metabolism , Animals , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Movement/genetics , Cells, Cultured , Epidermal Cells , Epidermis/embryology , Epidermis/metabolism , Immunohistochemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Melanocytes/cytology , Mice , Mice, Transgenic , Microscopy, Fluorescence , Neural Crest/cytology , Neural Crest/embryology , Neuroepithelial Cells/cytology , Neuroepithelial Cells/metabolism , SOXB1 Transcription Factors/genetics , Schwann Cells/cytology , Skin/cytology , Skin/embryology
7.
Nat Methods ; 8(5): 409-12, 2011 May.
Article in English | MEDLINE | ID: mdl-21460823

ABSTRACT

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.


Subject(s)
Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/cytology , Asian People/genetics , Electroporation , Gene Expression Profiling , Gene Frequency , Genetic Vectors , HLA Antigens/genetics , Humans , Induced Pluripotent Stem Cells/immunology , Induced Pluripotent Stem Cells/metabolism , Plasmids/genetics , Tissue Donors
8.
Arch Oral Biol ; 55(9): 648-54, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20630496

ABSTRACT

The hypoxia condition was expected to be suitable for the establishment and maintenance of human dental pulp cells (hDPCs), because they reside in a low-oxygen environment in vivo. Therefore, we presently examined the effects of hypoxia on the proliferation and differentiation of hDPCs in vitro. hDPCs grown under 3% O(2) showed a significantly higher proliferation rate than those under 21% O(2). Then, we prepared hypoxic cultures of hDPCs from older patients' teeth having inflammation and succeeded in recovering and expanding a small number of hDPCs. These cells were confirmed to have capability for osteo/odontogenic differentiation. Hypoxia suppressed the osteo/odontogenic differentiation of hDPCs in vitro and increased the number of cells expressing STRO-1, an early mesenchymal stem cell marker. This simple method will increase the possibility to obtain living hDPCs from damaged and/or aged tissues, from which it is ordinarily difficult to isolate living stem cells with differentiation capability.


Subject(s)
Adult Stem Cells/cytology , Dental Pulp/cytology , Epithelial Cells/cytology , Hypoxia/pathology , Mesenchymal Stem Cells/cytology , Adolescent , Adult Stem Cells/metabolism , Aged , Antigens, Surface/biosynthesis , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Cells, Cultured , Dental Pulp/metabolism , Epithelial Cells/metabolism , Extracellular Matrix Proteins/biosynthesis , Flow Cytometry , Humans , Hypoxia/metabolism , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Middle Aged , Osteocalcin/biosynthesis , Phosphoproteins/biosynthesis , Polymerase Chain Reaction , Sialoglycoproteins/biosynthesis
9.
FEBS Lett ; 581(27): 5321-6, 2007 Nov 13.
Article in English | MEDLINE | ID: mdl-17977532

ABSTRACT

POEM, also called nephronectin, is an extracellular matrix protein that is considered to play a critical role as an adhesion molecule in the development and functioning of various tissues, such as kidneys and bones. In the present study, we examined the molecular mechanism of POEM gene expression, and found that transforming growth factor-beta (TGF-beta) strongly inhibited POEM expression in the mouse osteoblastic cell line, MC3T3-E1. TGF-beta-induced decrease of POEM expression occurred in both time- and dose-dependent manners through the activation of TGF-beta receptor I and extracellular signal-regulated kinase/c-Jun N-terminal kinase pathways.


Subject(s)
Extracellular Matrix Proteins/genetics , MAP Kinase Signaling System/drug effects , Osteoblasts/drug effects , Osteoblasts/metabolism , Transforming Growth Factor beta1/pharmacology , 3T3 Cells , Animals , Base Sequence , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/pharmacology , Cells, Cultured , Down-Regulation/drug effects , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Models, Biological , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , Recombinant Proteins/pharmacology , Transforming Growth Factor beta/pharmacology
10.
Clin Calcium ; 16(8): 1374-79, 2006 Aug.
Article in Japanese | MEDLINE | ID: mdl-16883047

ABSTRACT

Notch is a signaling molecule which plays a critical role in the determination of multiple cellular differentiation pathways and morphogenesis in various biological systems, such as neurogenesis, immune system, and hematopoiesis. However, roles of Notch signaling in osteo/chondrogenesis have not been well studied. We will present our recent progress in investigating roles of Notch signaling in chondrogenesis using in vitro chondrogenic system. We will also discuss about the recent reports which used conditional knockout mice to investigate roles of Notch signaling molecules in vivo .


Subject(s)
Chondrogenesis/physiology , Receptors, Notch/physiology , Signal Transduction , Animals , Cells, Cultured , Mice
11.
J Bone Miner Metab ; 24(3): 191-8, 2006.
Article in English | MEDLINE | ID: mdl-16622731

ABSTRACT

Notch signaling is an evolutionarily conserved mechanism that plays a critical role in the determination of multiple cellular differentiation pathways and morphogenesis during embryogenesis. The limb bud high-density culture is an established model that recapitulates mesenchymal condensation and chondrocyte differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) showed that Notch and its related genes were expressed in such cultures on day 1 and reached a peak between day 3 and day 5, when cell condensation and nodule formation were initiated. Immunohistochemical experiments revealed that the expression of Notch1 was initially localized within the nodules and shifted to their peripheral region as the cell differentiation progressed. We disrupted Notch signaling by using a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), to analyze the function of Notch signaling in the culture system. The blocking of Notch signaling by DAPT apparently promoted the initiation of prechondrogenic condensation and fusion of the nodules, and such an effect was reversed by exogenous expression of the Notch cytoplasmic domain. DAPT treatment also induced chondrogenic markers and bone morphogenetic protein (BMP)-related molecules, including type II collagen, Sox9, GDF5, and Id1. These observations imply that the Notch signal may have an important role in chondrogenic differentiation by negatively regulating the initiation of prechondrogenic condensation and nodule formation.


Subject(s)
Receptors, Notch/physiology , Signal Transduction/physiology , Animals , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins/biosynthesis , Cell Differentiation , Cells, Cultured , Chondrocytes/physiology , Dipeptides/pharmacology , Growth Differentiation Factor 5 , Limb Buds/cytology , Mice , Mice, Inbred C57BL , Receptor, Notch1/biosynthesis , Receptors, Notch/biosynthesis , Signal Transduction/drug effects
12.
Bone ; 36(5): 877-83, 2005 May.
Article in English | MEDLINE | ID: mdl-15820146

ABSTRACT

Osteocytes are surrounded by hard bone matrix. Therefore, it has not previously been possible to demonstrate the real architecture of the osteocyte network in bone. We previously reported that it is possible to observe osteocytes in bone by labeling the cells with fluorescence and using confocal laser scanning (CLS) microscopy. In this study, we for the first time conducted an extensive analysis of the morphology and morphometry of the three-dimensional (3D) osteocyte structure using three-dimensionally reconstructed fluorescent images. Sixteen-day-old embryonic chick calvariae were stained with fluorescently labeled phalloidin and observed using a confocal laser scanning microscope. Morphometry of osteocytes in the calvaria was analyzed using extensive three-dimensional reconstructing software IMARIS, process length measuring software NEURON TRACER and cell surface area-/cell volume-analyzing software SURPASS. From the IMARIS-derived images, we found that the average of 10 osteocytes is 52.7 +/- 5.7 processes, and the point-to-point distance between centers of the osteocytes was 24.1 +/- 2.8 microm. In addition, we could calculate that each osteocyte spans an average of 4180 +/- 673 microm3 of bone volume. NEURON TRACER showed that the length of osteocyte processes was 0.26 +/- 0.02 microm per 1 microm3 bone compartment. In addition, SURPASS indicated that the surface area of osteocytes was 0.36 +/- 0.03 microm2 per 1 microm3 bone compartment and that the volume ratio of osteocyte cell body to bone compartment was 9.42% +/- 1.18%. Together, the average total length of the processes, the average surface area, and the average volume of one osteocyte were 1070 +/- 145 microm, 1509 +/- 113 microm2, and 394 +/- 49 microm3, respectively. It is possible to reconstruct the real architecture of the osteocyte network and obtain morphometric data from fluorescently labeled osteocytes in chick calvaria.


Subject(s)
Osteocytes/cytology , Skull/cytology , Animals , Chick Embryo , Microscopy, Confocal
14.
J Bone Miner Metab ; 23(1): 1-7, 2005.
Article in English | MEDLINE | ID: mdl-15616887

ABSTRACT

Bone is a complex system with functions including those of adaptation and repair. To understand how bone cells can create a structure adapted to the mechanical environment, we propose a simple bone remodeling model based on a reaction-diffusion system influenced by mechanical stress. Two-dimensional bone models were created and subjected to mechanical loads. The conventional finite element method (FEM) was used to calculate stress distribution. A stress-reactive reaction-diffusion model was constructed and used to simulate bone remodeling under mechanical loads. When an external mechanical stress was applied, stimulated bone formation and subsequent activation of bone resorption produced an efficient adaptation of the internal shape of the model bone to a given stress, and demonstrated major structures of trabecular bone seen in the human femoral neck. The degree of adaptation could be controlled by modulating the diffusion constants of hypothetical local factors. We also tried to demonstrate the deformation of bone structure during osteoporosis by the modulation of a parameter affecting the balance between formation and resorption. This simple model gives us an insight into how bone cells can create an architecture adapted to environmental stress, and will serve as a useful tool to understand both physiological and pathological states of bone based on structural information.


Subject(s)
Bone Remodeling/physiology , Bone and Bones/anatomy & histology , Bone and Bones/cytology , Computer Simulation , Models, Biological , Diffusion , Osteogenesis
15.
J Bone Miner Metab ; 21(6): 344-52, 2003.
Article in English | MEDLINE | ID: mdl-14586790

ABSTRACT

Notch is a transmembrane protein involved in cell fate determination. In the present study, we observed temporally and spatially restricted expression of Notch1 in developing cartilage. Notch1 was localized starting from the mesenchymal condensation stage of embryonic mouse forelimbs. Interestingly, although localization could not be detected in the proliferating chondrocytes, obvious immunoreactivity indicating its expression was retained in the perichondrial region. Next, we investigated the expression of Notch1 and related molecules in a chondrogenic cell line, ATDC5 cells. Notch1, Delta-like (Dll)1, Deltex2, and Deltex3 were coexpressed after 6-day insulin treatment. Expression of Hairy and Enhancer of split homologue (HES)-1 followed thereafter. These results suggest that Notch may have a role in the early stage of chondrogenesis. To assess the effect of Notch activation, we cultured ATDC5 cells with a myeloma clone constitutively expressing Dll1, a ligand of Notch. We also used an adenovirus vector to express the constitutively active Notch1 intracellular domain (NIC). Activating either the endogenous or exogenous Notch receptor dramatically inhibited chondrogenic cell differentiation of ATDC5 cells, as assessed by Alcian blue staining of the cells and chondrocyte differentiation markers. Last, we investigated the effect of NIC on the proliferation of the ATDC5 cells. Expression of NIC by the adenovirus strongly suppressed thymidine incorporation. These results indicate that Notch is expressed in the initial stage of chondrogenic cell differentiation and has a strong inhibitory effect on both differentiation and proliferation of the cells when activated. The expression of Notch decreases as chondrogenic differentiation proceeds; however, a population of the cells with sustained expression of Notch1 become perichondrial cells. Considering that the perichondrium acts as a stem cell source of osteoblasts and chondrocytes, Notch1 may have a role in the formation of these cells by suppressing both differentiation and proliferation.


Subject(s)
Chondrocytes/cytology , Membrane Proteins/metabolism , Adenoviridae/genetics , Adenoviridae/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors , Cartilage/cytology , Cell Differentiation , Cell Division , Cell Line , Chondrocytes/metabolism , Genetic Vectors , Homeodomain Proteins/metabolism , Membrane Proteins/genetics , Mice , Receptors, Notch , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Thymidine/metabolism , Transcription Factor HES-1
16.
J Bone Miner Res ; 17(2): 231-9, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11811553

ABSTRACT

Notch is a transmembrane protein that plays a critical role in the determination of cellular differentiation pathways. Although its importance in the development of mesenchymal tissues has been suggested, its role in skeletal tissues has not been well investigated. Northern blot experiments showed the expression of Notch1 in MC3T3-E1 osteoblastic cells at early differentiation stages. When a Notch1 cytoplasmic domain (Notch-IC [NIC]) delivered by an adenovirus vector was expressed in osteoblastic MC3T3-E1 cells, a significant increase in calcified nodule formation was observed in long-term cultures. Activation of endogenous Notch in MC3T3-E1 by coculturing them with Delta-like-1 (Dll1)-expressing myeloma cells also resulted in a stimulation of calcified nodule formation. Not only affecting nodule formation, Notch activation also had effects on osteoblastic differentiation of multipotent mesenchymal cells. Osteoblastic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2 (BMP-2) was significantly stimulated, whereas adipogenic differentiation was suppressed strongly, resulting in a dominant differentiation of osteoblastic cells. NIC expression in primary human bone marrow mesenchymal stem cells (hMSCs) also induced both spontaneous and stimulated osteoblastic cell differentiation. These observations suggest that osteoblastic cell differentiation is regulated positively by Notch and that Notch could be a unique and interesting target molecule for the treatment of osteoporosis.


Subject(s)
Cell Differentiation , Membrane Proteins/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Transcription Factors , Transforming Growth Factor beta , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/pharmacology , Cells, Cultured , Cytoplasm , Green Fluorescent Proteins , Humans , Intracellular Signaling Peptides and Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Proteins/genetics , Mice , Osteoblasts/drug effects , Receptor, Notch1 , Receptor, Notch2 , Receptors, Cell Surface , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
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