RESUMO
Primary cultured odontoblasts rapidly lose their tissue-specific phenotype. To identify transcription factors (TF) that are important for the maintenance of the odontoblast phenotype, primary cultures of C57BL/6 J mouse dental mesenchymal cells (DMC) were isolated, and expression of TF and odontoblast marker genes in cells immediately after isolation and 2 days after culture were comprehensively evaluated and compared using RNA-sequencing (RNA-seq). The expression of odontoblast markers in mouse dental mesenchymal cells decreased rapidly after isolation. In addition, the expression of Hedgehog-related, Notch-related, and immediate- early gene (IEG)-related transcription factors significantly decreased. Forced expression of these genes in lentiviral vectors, together with fibroblast growth factor 4 (FGF4), fibroblast growth factor 9 (FGF9), and the Wnt pathway activator CHIR99021, significantly induced the expression of odontogenic marker genes. These results indicate, for the first time, that Notch signaling and early genes may be important for maintaining odontoblast cultures. Furthermore, simultaneous stimulation of FGF, Wnt, Hedgehog, Notch pathways, and IEG transcription factors cooperatively promoted the maintenance of the odontoblast phenotype. These results suggest that the Hedgehog and Notch signaling pathways may play an important role in maintaining odontoblast phenotypes, in addition to FGF and Wnt signaling.
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Mechanical stimuli serve as crucial regulators of bone mass, promoting bone formation. However, the molecular mechanisms governing how mesenchymal stem cells (MSCs) respond to mechanical cues during their differentiation into osteogenic cells remain elusive. In this study, we found that cyclic stretching enhances MSC proliferation but does not increase the expression of osteoblast-related genes. We further revealed that this proliferative effect is mediated by fibroblast growth factor 2 (FGF-2), synthesized by MSCs in response to mechanical stress. Cell proliferation induced by cyclic stretching was inhibited upon the addition of either U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), or early growth response 1 (EGR1)-targeting small-hairpin RNA (shRNA), indicating the involvement of the extracellular signal-regulated kinase (ERK)/EGR1 signaling pathway. Osteoblast differentiation, evaluated through ALP activity, osteoblast-related gene expression, and mineralization, was stimulated by recombinant human FGF-2 (rhFGF-2) when applied during the proliferation phase, but not when applied during the differentiation stage alone. Our results suggest that FGF-2 indirectly promotes osteoblast differentiation as a downstream effect of stimulating cell proliferation. For the first time, we demonstrate that cyclic stretching induces MSCs to produce FGF-2, which in turn encourages cell proliferation through an autocrine/paracrine mechanism, consequently leading to osteoblast differentiation.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Humanos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Estresse Mecânico , Diferenciação Celular , Proliferação de Células , Osteoblastos/metabolismoRESUMO
The hedgehog (Hh) family consists of numerous signaling mediators that play important roles at various stages of development. Thus, the Hh pathway is essential for bone tissue development and tumorigenesis. Gorlin syndrome is a skeletal and tumorigenic disorder caused by gain-of-function mutations in Hh signaling. In this review, we first present the phenotype of Gorlin syndrome and the relationship between genotype and phenotype in bone and craniofacial tissues, including the causative gene as well as other Hh-related genes. Next, the importance of new diagnostic methods using next-generation sequencing and multiple gene panels will be discussed. We summarize Hh-related genetic disorders, including cilia disease, and the genetics of Hh-related bone diseases.
Assuntos
Síndrome do Nevo Basocelular , Doenças Ósseas , Humanos , Proteínas Hedgehog/genética , Mutação , Osso e Ossos , CarcinogêneseRESUMO
The development of next-generation sequencing (NGS) has dramatically increased the speed and volume of genetic analysis. Furthermore, the range of applications of NGS is rapidly expanding to include genome, epigenome (such as DNA methylation), metagenome, and transcriptome analyses (such as RNA sequencing and single-cell RNA sequencing). NGS enables genetic research by offering various sequencing methods as well as combinations of methods. Bone tissue is the most important unit supporting the body and is a reservoir of calcium and phosphate ions, which are important for physical activity. Many genetic diseases affect bone tissues, possibly because metabolic mechanisms in bone tissue are complex. For instance, the presence of specialized immune cells called osteoclasts in the bone tissue, which absorb bone tissue and interact with osteoblasts in complex ways to support normal vital functions. Moreover, the many cell types in bones exhibit cell-specific proteins for their respective activities. Mutations in the genes encoding these proteins cause a variety of genetic disorders. The relationship between age-related bone tissue fragility (also called frailty) and genetic factors has recently attracted attention. Herein, we discuss the use of genomic, epigenomic, transcriptomic, and metagenomic analyses in bone genetic disorders.
Assuntos
Doenças Ósseas , Osso e Ossos , Humanos , Doenças Ósseas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Osteoblastos , OsteoclastosRESUMO
The purpose of this study was to investigate whether fibroblast growth factor 4 (FGF4) and FGF9 are active in dentin differentiation. Dentin matrix protein 1 (Dmp1) -2A-Cre transgenic mice, which express the Cre-recombinase in Dmp1-expressing cells, were crossed with CAG-tdTomato mice as reporter mouse. The cell proliferation and tdTomato expressions were observed. The mesenchymal cell separated from neonatal molar tooth germ were cultured with or without FGF4, FGF9, and with or without their inhibitors ferulic acid and infigratinib (BGJ398) for 21 days. Their phenotypes were evaluated by cell count, flow cytometry, and real-time PCR. Immunohistochemistry for FGFR1, 2, and 3 expression and the expression of DMP1 were performed. FGF4 treatment of mesenchymal cells obtained promoted the expression of all odontoblast markers. FGF9 failed to enhance dentin sialophosphoprotein (Dspp) expression levels. Runt-related transcription factor 2 (Runx2) was upregulated until day 14 but was downregulated on day 21. Compared to Dmp1-negative cells, Dmp1-positive cells expressed higher levels of all odontoblast markers, except for Runx2. Simultaneous treatment with FGF4 and FGF9 had a synergistic effect on odontoblast differentiation, suggesting that they may play a role in odontoblast maturation.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Fator 4 de Crescimento de Fibroblastos , Fator 9 de Crescimento de Fibroblastos , Odontoblastos , Animais , Camundongos , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Camundongos Transgênicos , Odontoblastos/metabolismo , Fator 9 de Crescimento de Fibroblastos/genética , Fator 9 de Crescimento de Fibroblastos/metabolismoRESUMO
This study aimed to assess the combined application of two biomaterials, a selfassembling peptide hydrogel (SPH) and an atelocollagen sponge (ACS). The ACS was combined with SPH (PuraMatrixâ or PanaceaGelâ) and its osteogenic effects on mouse osteoblastic cell line MC3T3 then evaluated. Each type of SPH was successfully incorporated into the ACS. The MC3T3 cells showed uniform distribution within the scaffold. No necrotic cells were observed throughout the experimental procedures. When the SPH was combined with the ACS, the MC3T3 cells differentiated toward the osteo-lineage, expressing Alp, Runx2, Osx, Bsp, and Oc. PanaceaGelâ exhibited a stronger osteogenic effect on the cells than PuraMatrixâ.
Assuntos
Colágeno , Hidrogéis , Camundongos , Animais , Peptídeos/farmacologia , Diferenciação Celular , Osteogênese , OsteoblastosRESUMO
Hajdu-Cheney syndrome (HCS) is a rare autosomal dominant manifestation of a congenital genetic disorder caused by a mutation in the NOTCH2 gene. NOTCH signaling has variations from NOTCH 1 to 4 and maintains homeostasis by determining and regulating the proliferation and differentiation of various cells. In HCS, the over-accumulated NOTCH2 causes abnormal bone resorption due to its continuous excessive signaling. HCS is characterized by progressive bone destruction, has complex wide-range clinical manifestations, and significantly impacts the patient's quality of life. However, no effective treatment has been established for HCS to date. There are genetic variants of NOTCH2 that have been reported in the ClinVar database of the U.S. National Institutes of Health. In total, 26 mutant variants were detected based on the American College of Medical Genetics and Genomics (ACMC). To date, there has been no comprehensive compilation of HCS mutations. In this review, we provide the most comprehensive list possible of HCS variants, nucleotide changes, amino acid definitions, and molecular consequences reported to date, following the ACMC guidelines.
Assuntos
Síndrome de Hajdu-Cheney , Aminoácidos/genética , Pesquisa em Genética , Síndrome de Hajdu-Cheney/genética , Síndrome de Hajdu-Cheney/metabolismo , Humanos , Mutação , Nucleotídeos , Qualidade de VidaRESUMO
Fibroblast growth factor 8 (FGF8) is known to be a potent stimulator of canonical Wnt/ß-catenin activity, an essential factor for tooth development. In this study, we analyzed the effects of co-administration of FGF8 and a CHIR99021 (GSK3ß inhibitor) on differentiation of dental mesenchymal cells into odontoblasts. Utilizing Cre-mediated EGFP reporter mice, dentin matrix protein 1 (Dmp1) expression was examined in mouse neonatal molar tooth germs. At birth, expression of Dmp1-EGFP was not found in mesenchymal cells but rather epithelial cells, after which Dmp1-positive cells gradually emerged in the mesenchymal area along with disappearance in the epithelial area. Primary cultured mesenchymal cells from neonatal tooth germ specimens showed loss of Dmp1-EGFP positive signals, whereas addition of Wnt3a or the CHIR99021 significantly regained Dmp1 positivity within approximately 2 weeks. Other odontoblast markers such as dentin sialophosphoprotein (Dspp) could not be clearly detected. Concurrent stimulation of primary cultured mesenchymal cells with the CHIR99021 and FGF8 resulted in significant upregulation of odonto/osteoblast proteins. Furthermore, increased expression levels of runt-related transcription factor 2 (Runx2), osterix, and osteocalcin were also observed. The present findings indicate that coordinated action of canonical Wnt/ß-catenin and FGF8 signals is essential for odontoblast differentiation of tooth germs in mice.
Assuntos
Células-Tronco Mesenquimais , Odontoblastos , Animais , Diferenciação Celular , Fator 8 de Crescimento de Fibroblasto/metabolismo , Camundongos , Odontoblastos/metabolismo , OsteoblastosRESUMO
We investigated whether BMP4, FGF8, and/or WNT3a on neural crest-like cells (NCLC) derived from mouse induced pluripotent stem (miPS) cells will promote differentiation of odontoblasts-like cells. After the miPS cells matured into embryonic body (EB) cells, they were cultured in a neural induction medium to produce NCLC. As the differentiation of NCLC were confirmed by RT-qPCR, they were then disassociated and cultured with a medium containing, BMP4, FGF8, and/or WNT3a for 7 and 14 days. The effect of these stimuli on NCLC were assessed by RT-qPCR, ALP staining, and immunocytochemistry. The cultured EB cells presented a significant increase of Snai1, Slug, and Sox 10 substantiating the differentiation of NCLC. NCLC stimulated with more than two stimuli significantly increased the odontoblast markers Dmp-1, Dspp, Nestin, Alp, and Runx2 expression compared to control with no stimulus. The expression of Dmp-1 and Dspp upregulated more when FGF8 was combined with WNT3a. ALP staining was positive in groups containing BMP4 and fluorescence was observed in immunocytochemistry of the common significant groups between Dmp-1 and Dspp. After stimulation, the cell morphology demonstrated a spindle-shaped cells with long projections resembling odontoblasts. Simultaneous BMP4, FGF8, and WNT3a stimuli significantly differentiated NCLC into odontoblast-like cells.
Assuntos
Proteína Morfogenética Óssea 4 , Fator 8 de Crescimento de Fibroblasto , Células-Tronco Pluripotentes Induzidas , Odontoblastos , Animais , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Células Cultivadas , Fator 8 de Crescimento de Fibroblasto/farmacologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Crista Neural , Odontoblastos/metabolismo , Proteína Wnt3A/farmacologiaRESUMO
Cleidocranial dysplasia (CCD) is a hereditary disorder associated with skeletal dysplasia and dental abnormalities. CCD arises from heterozygous loss of function mutations in the Runt-related transcription factor 2 (RUNX2) gene. Osteoporosis is often observed in CCD patients and conventional vitamin D supplementation is recommended. However, sufficient evidences have not been presented yet. This study investigated the role of RUNX2 in osteoblastic differentiation and sought to identify potential target genes for the treatment of osteoporosis associated with CCD, using induced pluripotent stem cell (iPSC) technology. We successfully established Runx2-/-, Runx2+/- and wild-type miPSCs from litter-matched mice and found poor Vdr expression in Runx2-/-cells. Significant down-regulation of osteoblastic differentiation in Runx2-/- miPSCs was observed. Gene expression array revealed unexpected results such as remarkable increase of Rankl expression and decrease of Vdr in Runx2-/- cells. Insufficient response to vitamin D in Runx2-/- cells was also observed. Our results suggest that RUNX2 functions as a regulator of Rankl and Vdr and thereby controls bone density. These findings also suggest that conventional vitamin D supplementation may not be as effective as previously expected, in the treatment of osteoporosis associated with CCD, and that inhibiting RANKL function might be worth considering as an alternative treatment strategy.
Assuntos
Displasia Cleidocraniana , Subunidade alfa 1 de Fator de Ligação ao Core , Células-Tronco Pluripotentes Induzidas , Osteoporose , Vitamina D , Animais , Diferenciação Celular , Displasia Cleidocraniana/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Osteoporose/genética , Vitamina D/farmacologiaRESUMO
Serum serves as a source of rich nutrients during in vitro cell culture, facilitating cell adhesion, growth, and differentiation. When culturing stem cells for transplantation, however, it must be remembered that such culture medium may contain substances potentially harmful to the proposed recipient and may even induce cellular damage. The purpose of this study was to determine whether KnockOut Serum Replacement (KSR), a chemically defined medium supplement, enhanced in vitro differentiation of induced pluripotent stem cells into odontoblasts. Cranial neural crest cells, precursors of odontoblasts, were generated from mouse-induced pluripotent stem cells. They were then cultured in serum-free Dulbecco's modified Eagle's/F12 medium containing fibroblast growth factor 8 with or without KSR. The cells cultured with KSR showed strong proliferation, acquired a spindle-like morphology, and connected with the surrounding cells. KnockOut Serum Replacement also boosted expression of odontoblast markers as measured by qRT-PCR, and increased dentin sialoprotein as assessed by immunostaining. These results confirmed that mouse-induced pluripotent stem cells differentiated into odontoblasts under serum-free conditions, and that KSR enhanced the efficiency of this process.
Assuntos
Células-Tronco Pluripotentes Induzidas , Odontoblastos , Animais , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , CamundongosRESUMO
Gorlin syndrome (GS) is an autosomal dominant genetic disorder involving Patched 1 (PTCH1) mutations. The PTCH1 is a receptor as well as an inhibitor of hedgehog (Hh) to sequester downstream Hh pathway molecules called Smoothened (SMO). PTCH1 mutations causes a variety of GS conditions including falx calcification, odontogenic keratocytes and basal cell carcinomas (BCC). Because PTCH1 is a major driver gene of sporadic BCC, GS patients are characteristically prone to BCC. In order to elucidate the pathological mechanism of BCC-prone GS patients, we investigated keratinocytes derived from GS patient specific iPS cells (G-OFiPSCs) which were generated and reported previously. We found that keratinocytes derived from G-OFiPSCs (GKCs) have increased expression of Hh target molecules. GKCs were irradiated and those cells showed high resistance to UV induced apoptosis. BCL2, known as anti-apoptotic molecule as well as Hh target, significantly increased in GKCs. Several molecules involved in DNA repair, cell cycle control, senescence, and genotoxic stress such as TP53, BRCA1 and GADD45A increased only in GKCs. GKCs are indicated to be resistant to UV irradiation by upregulating molecules which control DNA repair and genotoxic even under DNA damage caused by UV. The anti-apoptotic properties of GKCs may contribute BCC.
Assuntos
Síndrome do Nevo Basocelular/metabolismo , Ciclo Celular , Reparo do DNA , Queratinócitos/metabolismo , Receptor Patched-1/genética , Raios Ultravioleta , Apoptose , Povo Asiático , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Síndrome do Nevo Basocelular/genética , Síndrome do Nevo Basocelular/fisiopatologia , Carcinoma Basocelular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Queratinócitos/fisiologia , Queratinócitos/efeitos da radiação , Mutação , Transdução de Sinais , Receptor Smoothened/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in the calcification sites of the skeletal tissue. It promotes hydroxyapatite crystal formation by degrading inorganic pyrophosphate (PPi) and increasing inorganic phosphate (Pi) concentration. However, abnormalities in Alpl-/- mouse-derived osteoblasts are poorly understood, and the involvement of TNAP in osteoblast differentiation remains unclear. Therefore, in this study, we aimed to investigate the precise role of TNAP in osteoblast differentiation. TNAP inhibition by levamisole, a reversible TNAP inhibitor, suppressed the expression of osteoblast differentiation marker genes in wild-type osteoblastic cells. Alpl overexpression increased the expression of master osteoblast transcription factor genes runt-related transcription factor 2 (Runx2) and Sp7 and the mature osteoblast and osteocyte marker genes, bone γ-carboxyglutamate protein 2 (Bglap2) and dentin matrix protein 1 (Dmp1), respectively in Alpl-deficient osteoblastic cells. TNAP regulated Runx2 expression, which in turn regulated the expression of all other osteoblast markers, except Dmp1. Dmp1 expression was independent of RUNX2 but was dependent on extracellular Pi concentration in Runx2-deficient osteogenic cells. These results suggest that TNAP functions as an osteogenic differentiation regulator either by regulating Runx2 expression or by controlling extracellular Pi concentration.
Assuntos
Fosfatase Alcalina/metabolismo , Diferenciação Celular , Osteogênese , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dura-Máter/citologia , Proteínas da Matriz Extracelular/metabolismo , Levamisol/farmacologia , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Fosfatos/farmacologia , Crânio/citologiaRESUMO
Thymic stromal lymphopoietin (TSLP) is a member of the IL-2 cytokine family, which is known to activate type 2 innate lymphoid cells, mast cells, and Th2 cells; this activation results in allergic inflammation and host defense against parasites. TSLP has also been shown to promote Th17-mediated immune responses, such as those observed in the development of rheumatoid arthritis; however, its role in osteoclastogenesis remains poorly understood. Here, we investigated the functional involvement of TSLP in RANKL-induced osteoclast differentiation from murine bone marrow-derived macrophages (BMMs). Both RANK- and RANK+ macrophages expressed TSLP receptor (TSLPR), while RANK+ osteoclast precursors maintained TSLPR expression after RANKL stimulation. TSLP stimulation led to inhibition of RANK-induced osteoclast differentiation in wild-type BMMs, but not Tslpr-/- BMMs; TSLP stimulation also led to suppression of osteoclastogenic gene expression (Nfatc1, Acp5, Mmp9, and Ctsk). These inhibitory effects of TSLP were significantly reduced following STAT1 inhibition. Finally, we found that LPS stimulation induced TSLP production in murine calvarial osteoblasts, but not BMMs. Together, these observations suggest that TSLP acts directly on osteoclast precursors to suppress osteoclastogenesis. Osteoblasts, along with other TSLP-producing cells, may therefore contribute to the inhibition of osteoclastogenesis under inflammatory conditions.
Assuntos
Citocinas/metabolismo , Osteogênese , Ligante RANK/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Linfopoietina do Estroma do TimoRESUMO
A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 (GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNASR201H-mutated (GNASR201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell-derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNASR201H-mutated induced pluripotent stem cells and GNASR201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNASR201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.
Assuntos
Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Mutação , Teratoma/patologia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Cromograninas/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos SCID , Teratoma/genéticaRESUMO
BACKGROUND/OBJECTIVES: PTH plays an important role in bone remodeling, and different actions have been reported depending on its administration method. iPSCs are promising as a cell source for regeneration of periodontal tissue due to their ability of proliferation and pluripotency. However, the effects of PTH on iPSCs remain mostly unknown. The purpose of this study was to investigate in vitro effects of parathyroid hormone (PTH) on osteoblastic differentiation of induced pluripotent stem cells (iPSCs) in a 3D culture model. MATERIALS AND METHODS: Following embryoid body (EB) induction from mouse iPSCs (miPSCs), dissociated cells (miPS-EB-derived cells) were seeded onto atelocollagen sponge (ACS) in osteoblast differentiation medium (OBM). Cell-ACS constructs were divided into three groups: continuous treatment with human recombinant PTH (1-34) (PTH-C), intermittent PTH treatment (PTH-I) or OBM control. To confirm the expression of PTH receptor-1(PTH1R), the expression of Pth1r and cAMP production over time were assessed. Real-time PCR was used to assess the expression of genes encoding osterix (Sp7), runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1), and osteocalcin (Bglap) at different time points. Mineralization was assessed by von Kossa staining. Histochemical staining was used to analyze alkaline phosphatase (ALP) activity, and immunolocalization of SP7 and BGLAP was analyzed by confocal laser scanning microscopy (CLSM). RESULTS: On days 7 and 14, expression of the Pth1r in miPS-EB-derived cells was increased in all groups. Production of cAMP, the second messenger of the PTH1R, tended to increase in the PTH-I group compared with PTH-C group on day 14. Expression of Col1a1 in the PTH-I group on day 14 was significantly higher than other groups. There was a time-dependent increase in the expression of Sp7 in all groups. On day 14, the expression level of Sp7 in the PTH-I group was significantly higher than other groups. In von Kossa staining, the PTH-I group showed higher level of staining compared with other groups on day 14, whereas the level was slightly attenuated in the PTH-C group. In histochemical staining, ALP-positive cells were significantly increased in the PTH-I group compared with other groups on day 14. In CLSM analysis, the numbers of SP7- and BGLAP-positive cells showed a gradual increase over time, and on day 14, a significantly greater SP7 expression was observed in the PTH-I group than other groups. CONCLUSION: These results suggested that the intermittent PTH treatment promotes osteoblastic differentiation and mineralization of miPSCs in the ACS scaffold.
Assuntos
Células-Tronco Pluripotentes Induzidas , Osteoblastos , Hormônio Paratireóideo , Fosfatase Alcalina , Animais , Diferenciação Celular , Humanos , Camundongos , Osteoblastos/fisiologia , Receptor Tipo 1 de Hormônio Paratireóideo/genéticaRESUMO
Gorlin syndrome is a skeletal disorder caused by a gain of function mutation in Hedgehog (Hh) signaling. The Hh family comprises of many signaling mediators, which, through complex mechanisms, play several important roles in various stages of development. The Hh information pathway is essential for bone tissue development. It is also the major driver gene in the development of basal cell carcinoma and medulloblastoma. In this review, we first present the recent advances in Gorlin syndrome research, in particular, the signaling mediators of the Hh pathway and their functions at the genetic level. Then, we discuss the phenotypes of mutant mice and Hh signaling-related molecules in humans revealed by studies using induced pluripotent stem cells.
Assuntos
Síndrome do Nevo Basocelular/genética , Testes Genéticos/métodos , Animais , Síndrome do Nevo Basocelular/diagnóstico , Síndrome do Nevo Basocelular/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Instabilidade Genômica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Receptores Patched/genética , Receptores Patched/metabolismoRESUMO
Cranial neural crest cells are multipotent cells that migrate into the pharyngeal arches of the vertebrate embryo and differentiate into various craniofacial organ derivatives. Therefore, migrating cranial neural crest cells are considered one of the most attractive candidate cell sources in regenerative medicine. We generated cranial neural crest like cell (cNCCs) using mouse-induced pluripotent stem cells cultured in neural crest-inducing medium for 14 days. Subsequently, we conducted RNA sequencing experiments to analyze gene expression profiles of cNCCs at different time points after induction. cNCCs expressed several neural crest specifier genes; however, some previously reported specifier genes such as paired box 3 and Forkhead box D3, which are essential for embryonic neural crest development, were not expressed. Moreover, ETS proto-oncogene 1, transcription factor and sex-determining region Y-box 10 were only expressed after 14 days of induction. Finally, cNCCs expressed multiple protocadherins and a disintegrin and metalloproteinase with thrombospondin motifs enzymes, which may be crucial for their migration.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Multipotentes/metabolismo , Crista Neural/metabolismo , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Animais , Biomarcadores/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Movimento Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Células-Tronco Multipotentes/citologia , Crista Neural/citologia , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Streptococcus mutans utilizes maltooligosaccharides, including maltose derived from human dietary starch. We recently reported that the glucose-phosphotransferase system (Glc-PTS) was also involved in the metabolism of glucose derived from intracellular maltooligosaccharides in S. mutans. The activity of the Glc-PTS was mediated by the mannose-(manLMN) and cellobiose-PTSs (celABRCD) in this organism. The purpose of this study was to identify which kind of glucose transporter was involved in this process. A celD, manLM, and glk triple mutant, cm6vU1, was constructed and its growth in maltose or glucose broth measured. When cm6vU1 cells were inoculated into a fresh glucose broth following prolonged incubation with glucose, their growth rate was greater than that in the initial inoculum. This suggested that an additional Glc-PTS was induced in these cells. To investigate this possibility, permeabilized S. mutans cells were constructed and Glc-PTS activity examined by photometrical assay method. Activity in the cells was higher in the secondary inocula than in the initial inocula. These results suggest that S. mutans possesses an additional as yet uncharacterized PTS transporter for glucose in addition to the mannose- and cellobiose-PTSs.