RESUMO
In this study, we examined the fundamental surface characteristics of a polyserotonin (pST) film, which is attractive as a bioelectrical and biocompatible interface of biosensors. The pST film can easily be modified on electrode materials such as Au by self-polymerization and electropolymerization. By a simple cytotoxicity test using nonadhesive living cells, we found that the pST film is biocompatible for culturing cells on it. This finding is also supported by the fact that the surface tension of the pST film is moderate for protein adsorptions. The pST film is thinner and smoother than a poly-dopamine film, the chemical structure of which is similar to that of the pST film, depending on the polymerization time, cycle, and temperature; thus, ST as the main monomer can facilitate the precise control of the thickness and roughness of functional polymer membranes on the nanometer order. In addition, the pST film is useful as a relatively insulative interface for preventing interfering species from approaching electrode surfaces without their nonspecific adsorption, depending on the surface charges of the pST film in solutions of different pHs. The formation of the pST film self-polymerized on electrode materials is derived from the adsorption of pST nanoparticles formed by oxidative polymerization under basic conditions; therefore, the process of pST film formation should be considered in the functionalization of the pST film as a bioelectrical interface that allows biomolecular recognition (e.g., molecularly imprinted polymer membrane) for its application to wearable and biocompatible biosensors.
Assuntos
Técnicas Biossensoriais , Polímeros , Adsorção , Eletrodos , Polimerização , Polímeros/químicaRESUMO
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
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
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
Proanthocyanidins, also known as condensed tannins and/or oligomeric flavonoids, occur in many edible plants and have various interesting biological activities. Previously, we reported a synthetic method for the preparation of various procyanidins in pure form and described their biological activities. Here, we describe the synthesis of procyanidin B1 acetylated analogs and discuss their inhibition activities against HeLa S3 cell proliferation. Surprisingly, the lower-unit acetylated procyanidin B1 strongly inhibited the proliferation of HeLa S3 cells. This molecule showed much stronger inhibitory activity than did epigallocatechin-3-O-gallate (EGCG), green tea polyphenol, and dimeric compounds that included EGCG as a unit. This result suggests that the phenolic hydroxyl groups of the upper-units in flavan-3-ols are important for their inhibitory activity against cancer cell proliferation and that a hydrophobic lower unit dimer enhances this activity.
Assuntos
Biflavonoides/síntese química , Catequina/síntese química , Proliferação de Células/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proantocianidinas/síntese química , Acetilação , Biflavonoides/química , Biflavonoides/farmacologia , Catequina/análogos & derivados , Catequina/química , Catequina/farmacologia , Células HeLa , Humanos , Neoplasias/patologia , Polímeros/química , Proantocianidinas/química , Proantocianidinas/farmacologia , Chá/químicaRESUMO
Chondroitin sulfate (CS) is a major microenvironmental molecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotoxicity is a crucial phase in many neuronal diseases, we examined the effect of various CS preparations on neuronal cell death induced by the excitotoxicity of glutamate analogs. CS preparations were added to cultured neurons before and after the administration of glutamate analogs. Then, the extents of both neuronal cell death and survival were estimated. Pre-administration of a highly sulfated CS preparation, CS-E, significantly reduced neuronal cell death induced by not only NMDA but also (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. Neither CS preparations other than CS-E nor other highly sulfated polysaccharides such as heparin and dextran sulfate exerted any neuroprotective effects. NMDA-induced current in neurons was not changed by pre-administration of CS-E, but the pattern of protein-tyrosine phosphorylation was changed. In addition, the elevation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E prevents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracellular signals and the suppression of caspase 3 activation are implicated in neuroprotection by CS-E.
Assuntos
Morte Celular/efeitos dos fármacos , Sulfatos de Condroitina/farmacologia , Degeneração Neural/tratamento farmacológico , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Ácido Caínico/farmacologia , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/toxicidade , Neocórtex/citologia , Degeneração Neural/metabolismo , Degeneração Neural/prevenção & controle , Neurotoxinas/toxicidade , Fosforilação , Polieletrólitos , Polímeros/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Tirosina/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
An oral infection harboring Fusobacterium species can gain entrance to the liver via hematogenous spread in the form of septic embolus, and can thereby cause abscesses. Such spread, described as Lemierre syndrome, is life threatening. We present such a case history of a man in his mid-40s, who presented with infection and Fusobacterium liver abscess with an acute fulminant disease course. The initial diagnosis was arrived at by ultrasound imaging and blood investigations. He was treated with antibiotics, ultrasound-guided liver abscess drainage, and extraction of the infected molar tooth. He was discharged 6 weeks after admission. To date, there have been no reports describing the ultrasound images of a Fusobacterium liver abscess in detail. Hence, we herein present the ultrasound images of a Fusobacterium liver abscess.
Assuntos
Infecções por Fusobacterium/diagnóstico por imagem , Abscesso Hepático/diagnóstico por imagem , Abscesso Hepático/etiologia , Fígado/diagnóstico por imagem , Ultrassonografia , Infecções por Fusobacterium/terapia , Humanos , Abscesso Hepático/terapia , Masculino , Pessoa de Meia-IdadeRESUMO
Surface-coated poly(catecholamine) (pCA) films have attracted attention as biomaterial interfaces owing to their biocompatible and physicochemical characteristics. In this paper, we report that pCA-film-coated electrodes are useful for potentiometric biosensing devices. Four different types of pCA film, l-dopa, dopamine, norepinephrine, and epinephrine, with thicknesses in the range of 7-27 nm were electropolymerized by oxidation on Au electrodes by using cyclic voltammetry. By using the pCA-film electrodes, the pH responsivities were found to be 39.3-47.7 mV/pH within the pH range of 1.68 to 10.01 on the basis of the equilibrium reaction with hydrogen ions and the functional groups of the pCAs. The pCA films suppressed nonspecific signals generated by other ions (Na+, K+, Ca2+) and proteins such as albumin. Thus, the pCA-film electrodes can be used in pH-sensitive and pH-selective biosensors. HeLa cells were cultivated on the surface of the pCA-film electrodes to monitor cellular activities. The surface potential of the pCA-film electrodes changed markedly because of cellular activity; therefore, the change in the hydrogen ion concentration around the cell/pCA-film interface could be monitored in real time. This was caused by carbon dioxide or lactic acid that is generated by cellular respiration and dissolves in the culture medium, resulting in the change of hydrogen concentration. pCA-film electrodes are suitable for use in biocompatible and pH-responsive biosensors, enabling the more selective detection of biological phenomena.
Assuntos
Técnicas Biossensoriais/métodos , Catecolaminas/química , Materiais Revestidos Biocompatíveis/química , Polímeros/química , Potenciometria/métodos , Técnicas Biossensoriais/instrumentação , Técnicas de Cultura de Células , Eletrodos , Ouro/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Potenciometria/instrumentação , Propriedades de SuperfícieRESUMO
BACKGROUND: Runt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models. METHODS: Two cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-derived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis. RESULTS: Mutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with severe combined immunodeficiency showed poor regeneration. However, reverted iPSCs improved the abnormal osteoblast differentiation which resulted in much better engraftment into the rat calvarial bone defect. CONCLUSIONS: Taken together, these results demonstrate that patient-specific iPSC technology can not only provide a useful disease model to elucidate the role of RUNX2 in osteoblastic differentiation but also raises the tantalizing prospect that reverted iPSCs might provide a practical medical treatment for CCD.
Assuntos
Regeneração Óssea/fisiologia , Displasia Cleidocraniana/terapia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Osteoblastos/citologia , Osteogênese/fisiologia , Osteoporose/terapia , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Células Cultivadas , Displasia Cleidocraniana/genética , Códon sem Sentido/genética , Modelos Animais de Doenças , Humanos , Fator 4 Semelhante a Kruppel , Osteoporose/patologia , RatosRESUMO
Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Causative mutations in several genes associated with the sonic hedgehog (SHH) signaling pathway, including PTCH1, have been identified in Gorlin syndrome patients. However, no definitive genotype-phenotype correlations are evident in these patients, and their clinical presentation varies greatly, often leading to delayed diagnosis and treatment. We generated iPSCs from four unrelated Gorlin syndrome patients with loss-of-function mutations in PTCH1 using the Sendai virus vector (SeVdp(KOSM)302). The patient-derived iPSCs exhibited basic iPSC features, including stem cell marker expression, totipotency, and the ability to form teratomas. GLI1 expression levels were greater in fibroblasts and patient-derived iPSCs than in the corresponding control cells. Patient-derived iPSCs expressed lower basal levels than control iPSCs of the genes encoding the Hh ligands Indian Hedgehog (IHH) and SHH, the Hh acetyltransferase HHAT, Wnt proteins, BMP4, and BMP6. Most of these genes were upregulated in patient-derived iPSCs grown in osteoblast differentiation medium (OBM) and downregulated in control iPSCs cultured in OBM. The expression of GLI1 and GLI2 substantially decreased in both control and patient-derived iPSCs cultured in OBM, whereas GLI3, SHH, and IHH were upregulated in patient-derived iPSCs and downregulated in control iPSCs grown in OBM. Activation of Smoothened by SAG in cells grown in OBM significantly enhanced alkaline phosphatase activity in patient-derived iPSCs compared with control iPSC lines. In summary, patient-derived iPSCs expressed lower basal levels than the control iPSCs of the genes encoding Hh, Wnt, and bone morphogenetic proteins, but their expression of these genes strongly increased under osteogenic conditions. These findings indicate that patient-derived iPSCs are hypersensitive to osteogenic induction. We propose that Hh signaling is constituently active in iPSCs from Gorlin syndrome patients, enhancing their response to osteogenic induction and contributing to disease-associated abnormalities.
Assuntos
Proteínas Hedgehog/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Osteogênese/genética , Transdução de Sinais/genética , Aciltransferases/genética , Adulto , Síndrome do Nevo Basocelular/genética , Síndrome do Nevo Basocelular/patologia , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 6/genética , Diferenciação Celular/genética , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Receptor Patched-1/genética , Proteínas Wnt/genética , Adulto Jovem , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
We recently reported a new method to purify the induced pluripotent stem (iPS)-derived osteoprogenitors (iPSop). In this paper, we optimized the procedure and characterized cells at each process step. We observed that 10 days of treatment with FGF-2, IGF-1 and TGF-ß (FIT) resulted in early-phase osteoblasts and 14 days of treatment resulted in late-phase osteoblasts. We found that treatment with 1,25(OH)2 vitamin D3 increased expression of osteocalcin and decreased expression of tissue-non-specific alkaline phosphatase and runt-related transcription factor 2 (RUNX2) in iPSop-day14 cells (cells treated with FIT for 14 days). Therefore, iPSop-day14 cells were promoted to mature osteoblasts by 1,25(OH)2 vitamin D3 treatment. In addition, we found that 1,25(OH)2 vitamin D3 treatment for 14 days enhanced not only mineralization but also expression of osteocyte markers, including dentin matrix protein-1 and fibroblast growth factor-23, in iPSop cells. Therefore, 1,25(OH)2 vitamin D3 is a potent promoter of osteoblast-osteocyte transition. The results of this study suggest that it is possible to evaluate both early- and late-phase osteoblasts and to apply cells to drug screening for anabolic drugs that stimulate bone formation.