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1.
J Appl Oral Sci ; 28: e20190215, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939521

RESUMO

OBJECTIVE: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). METHODOLOGY: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. RESULTS: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). CONCLUSION: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.


Assuntos
Indutores da Angiogênese/farmacologia , Compostos de Cálcio/farmacologia , Cerâmica/farmacologia , Materiais Restauradores do Canal Radicular/farmacologia , Silicatos/farmacologia , Células-Tronco/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Teste de Materiais , Neovascularização Fisiológica/efeitos dos fármacos , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/análise , Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Reprodutibilidade dos Testes , Estatísticas não Paramétricas , Germe de Dente/citologia , Germe de Dente/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos
2.
Arch Oral Biol ; 109: 104584, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31630006

RESUMO

OBJECTIVES: To investigate whether rutin could protect human periodontal ligament stem cells (hPDLSCs) from TNF-α induced damage to osteogenic differentiation in inflammatory environment and detect the underlying mechanism. MATERIALS AND METHODS: hPDLSCs were identified by flow cytometery. TNF-α was used to stimulate hPDLSCs to establish an inflammation model in vitro. Alkaline phosphatase (ALP) staining, ALP activity test, and Alizarin Red staining were used to detect the changes of osteogenic differentiation ability. The mRNA and protein levels of osteogenic genes were evaluated by RT-PCR and Western Blot. The expression of mTOR was also detected by Western Blot. RESULTS: hPDLSCs were positive to MSCs specific surface markers. The inflammatory environment in vitro could be established by stimulating hPDLSCs with TNF-α (20 ng/mL). TNF-α (20 ng/mL) could decrease the ALP activity and mineralization ability of hPDLSCs and down-regulate the expression of osteogenic genes in inflammatory environment. Moreover, rutin could affect TNF-α (20 ng/mL) induced damage to osteogenic differentiation of hPDLSCs in a dose-dependent manner, 10 µmol/L rutin could significantly reverse the damage caused by TNF-α. In addition, rutin inhibited TNF-α-activated mTOR signal transduction by inhibiting the phosphorylation of mTOR, similar to the effects of rapamycin(a specific mTOR inhibitor). CONCLUSIONS: Rutin could protect hPDLSCs from TNF-α induced damage to osteogenic differentiation in inflammatory environment, and rutin is expected to become a new candidate drug for the treatment of bone defect of periodontitis.


Assuntos
Osteogênese , Ligamento Periodontal/citologia , Rutina/farmacologia , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/efeitos adversos , Fosfatase Alcalina/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Inflamação , Células-Tronco/citologia
3.
Chem Biol Interact ; 316: 108919, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31846616

RESUMO

Ethanol (EtOH) is a recreationally ingested compound that is both teratogenic and carcinogenic in humans. Because of its abundant consumption worldwide and the vital role of stem cells in the formation of birth defects and cancers, delineating the effects of EtOH on stem cell function is currently an active and urgent pursuit of scientific investigation to explicate some of the mechanisms contributing to EtOH toxicity. Stem cells represent a primordial, undifferentiated phase of development; thus encroachment on normal physiologic processes of differentiation into terminal lineages by EtOH can greatly alter the function of progenitors and terminally differentiated cells, leading to pathological consequences that manifest as fetal alcohol spectrum disorders and cancers. In this review we explore the disruptive role of EtOH in differentiation of stem cells. Our primary objective is to elucidate the mechanisms by which EtOH alters differentiation-related gene expression and lineage specifications, thus modifying stem cells to promote pathological outcomes. We additionally review the effects of a reactive metabolite of EtOH, acetaldehyde (AcH), in causing both differentiation defects in stem cells as well as genomic damage that incites cellular aging and carcinogenesis.


Assuntos
Acetaldeído/farmacologia , Diferenciação Celular/efeitos dos fármacos , Etanol/farmacologia , Acetaldeído/metabolismo , Aldeído Oxirredutases/deficiência , Aldeído Oxirredutases/genética , Animais , Dano ao DNA/efeitos dos fármacos , Etanol/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
4.
Chem Biol Interact ; 316: 108931, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31874163

RESUMO

Bone defects caused by cancer surgery or trauma have a strong negative impact on human health. Treatment with cell and material-based complexes provides an alternative strategy for the regeneration of damaged bone tissue. The good physical properties and suitable biodegradability of a thermosensitive hydrogel has been shown to act as a valuable scaffold. Platelet derived growth factor BB (PDGFBB) is mainly secreted by platelets and promotes the migration and angiogenesis of mesenchymal stem cells (MSCs). Although PDGFBB is known to indirectly enhance bone repair in vivo, the effects of PDGFBB on stem cells from apical papilla (SCAPs) require further investigation. In our study, the proliferation of cells was investigated by the cell counting kit-8 and live/dead staining methods. The results indicated that PDGFBB promoted the proliferation of SCAPs. Real-time polymerase chain reaction and Western blot experiments were used to detect osteogenic genes and proteins. Moreover, calvarial defects were created in Sprague-Dawley rats and different complexes implanted. The results shown by micro-CT and hematoxylin and eosin analysis demonstrated that the hydrogel combined with lentiviral supernatant-green fluorescent protein-PDGFBB significantly improved new bone formation and mineralization compared with the other three groups. In summary, our research showed that a thermosensitive hydrogel can be used as a scaffold for 3D cell culture, and PDGFBB gene-modified SCAPs can improve bone formation in calvarial defects.


Assuntos
Becaplermina/metabolismo , Regeneração Óssea/fisiologia , Hidrogéis/química , Células-Tronco/metabolismo , Tecidos Suporte/química , Animais , Becaplermina/genética , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Papila Dentária/citologia , Hidrogéis/farmacologia , Masculino , Osteogênese , Polietilenoglicóis/química , Poliglactina 910/química , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Microtomografia por Raio-X
5.
Cell Physiol Biochem ; 53(6): 961-981, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31820856

RESUMO

BACKGROUND/AIMS: We assessed the effects of ticagrelor, aspirin and prasugrel, started 7days after myocardial ischemia-reperfusion injury on remodeling, inflammation and fibrosis in the rat. We examined whether ticagrelor can affect the number of progenitor cells in the border zone. Ticagrelor, started 24h after myocardial ischemia-reperfusion injury, attenuates the decrease in heart function and adverse remodeling, an effect which is blocked by aspirin. METHODS: Rats underwent 40min ischemia followed by reperfusion. Oral dosing with vehicle, ticagrelor (300mg/kg/d), aspirin (20mg/kg/d), their combination or prasugrel (15mg/kg/d) started 7days after infarction. Echocardiography was used to assess systolic function. Heart tissue were analyzed by rt-PCR, immunoblotting, ELISA and immunohistochemistry 2weeks after infarction. RESULTS: Both ticagrelor and aspirin attenuated the decrease in systolic function and remodeling, an effect that was blocked by their combination. Ticagrelor and aspirin attenuated the increase in ANP, BNP, collagen-I and collagen-III. Again, the effect was blocked by their combination. Ticagrelor increased c-Kit, Sca-1, Ki-67, CD34, attenuated the decrease in CD105 mRNA levels, and attenuated the increase in CD31, whereas aspirin increased Ki-67, suppressed the increase in CD31 and attenuated the decrease in CD105 mRNA levels. Prasugrel did not display any effects. CONCLUSION: Ticagrelor attenuated adverse remodeling and deterioration of left ventricular systolic function despite starting treatment after the myocardial ischemia-reperfusion injury is completed. Aspirin had similar effects; however, when combined with ticagrelor, the protective effects were significantly attenuated. Ticagrelor increased the levels of several markers of stem cells and regeneration, suggesting cardiac healing by recruiting regenerative cells into the infarct.


Assuntos
Apoptose/efeitos dos fármacos , Infarto do Miocárdio/patologia , Inibidores da Agregação de Plaquetas/farmacologia , Ticagrelor/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Aspirina/farmacologia , Aspirina/uso terapêutico , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Endoglina/genética , Endoglina/metabolismo , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Inibidores da Agregação de Plaquetas/uso terapêutico , Cloridrato de Prasugrel/farmacologia , Cloridrato de Prasugrel/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Ticagrelor/uso terapêutico , Função Ventricular Esquerda/efeitos dos fármacos
6.
PLoS One ; 14(12): e0226363, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856233

RESUMO

Cell-based tissue reconstruction is an important field of regenerative medicine. Stem and progenitor cells derived from tooth-associated tissues have strong regeneration potential. However, their in vivo application requires the development of novel scaffolds that will provide a suitable three-dimensional (3D) environment allowing not only the survival of the cells but eliciting their proliferation and differentiation. Our aim was to study the viability and differentiation capacity of periodontal ligament cells (PDLCs) cultured on recently developed biocompatible and biodegradable poly(aspartamide) (PASP)-based hydrogels. Viability and behavior of PDLCs were investigated on PASP-based hydrogels possessing different chemical, physical and mechanical properties. Based on our previous results, the effect of thiol group density in the polymer matrix on cell viability, morphology and differentiation ability is in the focus of our article. The chemical composition and 3D structures of the hydrogels were determined by FT Raman spectroscopy and Scanning Electron Microscopy. Morphology of the cells was examined by phase contrast microscopy. To visualize cell growth and migration patterns through the hydrogels, two-photon microscopy were utilized. Cell viability analysis was performed according to a standardized protocol using WST-1 reagent. PDLCs were able to attach and grow on PASP-based hydrogels. An increase in gel stiffness enhanced adhesion and proliferation of the cells. However, the highest population of viable cells was observed on the PASP gels containing free thiol groups. The presence of thiol groups does not only enhance viability but also facilitates the osteogenic direction of the differentiating cells. These cell-gel structures seem to be highly promising for cell-based tissue reconstruction purposes in the field of regenerative medicine.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Hidrogéis/farmacologia , Dente Serotino , Ligamento Periodontal/citologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Compostos de Sulfidrila/química , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Hidrogéis/química , Osteogênese/efeitos dos fármacos , Tecidos Suporte/química
7.
Res Vet Sci ; 126: 207-212, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31610471

RESUMO

To explore the effect of epigenetic modification on the differentiation of goat adipose-derived stem cells in vitro, we used two common epigenetic modification inhibitors, trichostatin A and vorinostat, to treat cashmere goat adipose-derived stem cells and induce adipocyte differentiation. The results showed that trichostatin A and vorinostat changed the relative amounts of H3K9 acetylation and dimethylation in the upstream sequence of PPARG, increased peroxisome proliferator-activated receptor gamma (PPARG) transcription before differentiation and then promoted adipocyte differentiation, and regulated the expression of adipocyte-specific genes. We conclude that adipocyte differentiation is regulated dynamically by different histone modifications. The areas of acetylation and demethylation changed by trichostatin A and vorinostat are the basis for further research on the mechanism of PPARG promoter to regulate adipocytes differentiation and provide research theroies for using adipose-derived stem cells as donor to produce transgenic animals to improve meat quality improvement.


Assuntos
Adipogenia/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Cabras/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Vorinostat/farmacologia , Acetilação/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipócitos/fisiologia , Animais , Metilação/efeitos dos fármacos , PPAR gama/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia
8.
Nat Commun ; 10(1): 4533, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586071

RESUMO

Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myeloma-induced bone disease that can be therapeutically targeted.


Assuntos
Doenças Ósseas/tratamento farmacológico , Proteínas Morfogenéticas Ósseas/metabolismo , Mieloma Múltiplo/complicações , Pirazóis/farmacologia , Pirimidinas/farmacologia , Células-Tronco/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas/etiologia , Doenças Ósseas/patologia , Medula Óssea/patologia , Receptores de Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fêmur/citologia , Fêmur/efeitos dos fármacos , Fêmur/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos , Mieloma Múltiplo/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/patologia , Tíbia/citologia , Tíbia/efeitos dos fármacos , Tíbia/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Int J Mol Sci ; 20(20)2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31600954

RESUMO

Periodontal disease is the main reason for tooth loss in adults. Tissue engineering and regenerative medicine are advanced technologies used to manage soft and hard tissue defects caused by periodontal disease. We developed a transforming growth factor-ß3/chitosan sponge (TGF-ß3/CS) to repair periodontal soft and hard tissue defects. We investigated the proliferation and osteogenic differentiation behaviors of primary human periodontal ligament stem cells (hPDLSCs) to determine the bioactivity and potential application of TGF-ß3 in periodontal disease. We employed calcein-AM/propidium iodide (PI) double labeling or cell membranes (CM)-Dil labeling coupled with fluorescence microscopy to trace the survival and function of cells after implantation in vitro and in vivo. The mineralization of osteogenically differentiated hPDLSCs was confirmed by measuring alkaline phosphatase (ALP) activity and calcium content. The levels of COL I, ALP, TGF-ßRI, TGF-ßRII, and Pp38/t-p38 were assessed by western blotting to explore the mechanism of bone repair prompted by TGF-ß3. When hPDLSCs were implanted with various concentrations of TGF-ß3/CS (62.5-500 ng/mL), ALP activity was the highest in the TGF-ß3 (250 ng/mL) group after 7 d (p < 0.05 vs. control). The calcium content in each group was increased significantly after 21 and 28 d (p < 0.001 vs. control). The optimal result was achieved by the TGF-ß3 (500 ng/mL) group. These results showed that TGF-ß3/CS promotes osteogenic differentiation of hPDLSCs, which may involve the p38 mitogen-activated protein kinase (MAPK) signaling pathway. TGF-ß3/CS has the potential for application in the repair of incomplete alveolar bone defects.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Quitosana , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/citologia , Células-Tronco/efeitos dos fármacos , Fator de Crescimento Transformador beta3/farmacologia , Biomarcadores , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quitosana/química , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Células-Tronco/citologia , Fator de Crescimento Transformador beta3/química
10.
Molecules ; 24(20)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623109

RESUMO

Nitric oxide (NO) is implicated in several biological processes, including cancer progression. At low concentrations, it promotes cell survival and tumor progression, and at high concentrations it causes apoptosis and cell death. Until now, the impact of NO donors has not been investigated on human endometrial tumors. Four cancer cell lines were exposed to different concentrations of DETA/NO for 24 to 120 h. The effects of DETA/NO on cell proliferation and invasion were determined utilizing MTS and Boyden chamber assays, respectively. The DETA/NO induced a dose and time-dependent reduction in cell viability by the activation of caspase-3 and cell cycle arrest at the G0/G1 phase that was associated with the attenuated expression of cyclin-D1 and D3. Furthermore, the reduction in the amount of CD133-expressing cancer stem-like cell subpopulation was observed following DETA/NO treatment of cells, which was associated with a decreased expression of stem cell markers and attenuation of cell invasiveness. To understand the mechanisms by which DETA/NO elicits anti-cancer effects, RNA sequencing (RNA-seq) was used to ascertain alterations in the transcriptomes of human endometrial cancer cells. RNA-seq analysis revealed that 14 of the top 21 differentially expressed genes were upregulated and seven were downregulated in endometrial cancer cells with DETA/NO. The genes that were upregulated in all four cell lines with DETA/NO were the tumor suppressors Ras association domain family 1 isoform A (RASSF1) and Cyclin-dependent kinase inhibitor 1A (CDKN1A). The expression patterns of these genes were confirmed by Western blotting. Taken together, the results provide the first evidence in support of the anti-cancer effects of DETA/NO in endometrial cancer.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Neoplasias do Endométrio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Proteínas Supressoras de Tumor/genética , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Óxido Nítrico/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor/metabolismo
11.
J Agric Food Chem ; 67(41): 11464-11473, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31532211

RESUMO

The intestinal epithelium is derived from intestinal stem cells (ISCs) and has direct contact with nutrients and toxins. However, whether methionine (Met) or a methionine hydroxyl analogue (2-hydroxy-4-(methylthio)butanoic acid (HMB)) can alleviate deoxynivalenol (DON)-induced intestinal injury remains unknown. Mice were treated orally with Met or HMB on days 1-11 and with DON on days 4-8. On day 12, the mice were sacrificed, and the jejunum was collected for crypt isolation and culture. Mouse enteroids were treated with DON and Met or HMB ex vivo. The results showed that Met and HMB increased the average daily feed intake and average daily gain of the mice. Met and HMB also improved the jejunal structure and barrier integrity and promoted ISC expansion, as indicated by the increased enteroid formation efficiency and area, under DON-induced injury conditions. In addition, DON-induced decreases in ISC activity were rescued Wnt/ß-catenin signaling reactivation by Met or HMB in vivo and ex vivo. Collectively, our findings reveal that Met and HMB alleviated DON-induced intestinal injury by improving ISC expansion and reactivating Wnt/ß-catenin signaling. Our study thus provides a nutritional intervention for intestinal diseases involving Wnt/ß-catenin signaling.


Assuntos
Enteropatias/tratamento farmacológico , Metionina/análogos & derivados , Metionina/administração & dosagem , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Tricotecenos/toxicidade , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Enteropatias/genética , Enteropatias/metabolismo , Enteropatias/fisiopatologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/lesões , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Células-Tronco/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
12.
Nat Commun ; 10(1): 4407, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562298

RESUMO

Understanding urothelial stem cell biology and differentiation has been limited by the lack of methods for their unlimited propagation. Here, we establish mouse urothelial organoids that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming cells expressing basal markers. Upon differentiation, multilayered organoids undergo reduced proliferation, decreased cell layer number, urothelial program activation, and acquisition of barrier function. Pharmacological modulation of PPARγ and EGFR promotes differentiation. RNA sequencing highlighted genesets enriched in proliferative organoids (i.e. ribosome) and transcriptional networks involved in differentiation, including expression of Wnt ligands and Notch components. Single-cell RNA sequencing (scRNA-Seq) analysis of the organoids revealed five clusters with distinct gene expression profiles. Together, with the use of γ-secretase inhibitors and scRNA-Seq, confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.


Assuntos
Diferenciação Celular/genética , Integrina alfa6/genética , Organoides/metabolismo , Receptores Notch/metabolismo , Células-Tronco/metabolismo , Urotélio/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Integrina alfa6/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Organoides/citologia , Organoides/efeitos dos fármacos , Receptores Notch/genética , Análise de Célula Única/métodos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Urotélio/citologia
13.
Mar Drugs ; 17(9)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505769

RESUMO

Intracellular reactive oxygen species (ROS) play an important role in the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). HSPCs are difficult to be expanded ex vivo while maintaining their stemness when they are exposed to oxidative damage after being released from the bone marrow. There have been efforts to overcome this limitation by using various cytokine cocktails and antioxidants. In this study, we investigated the effects of echinochrome A (Ech A)-a well-established and non-toxic antioxidant-on the ex vivo expansion of HSPCs by analyzing a CD34+ cell population and their biological functions. We observed that Ech A-induced suppression of ROS generation and p38-MAPK/JNK phosphorylation causes increased expansion of CD34+ cells. Moreover, p38-MAPK/JNK inhibitors SB203580 and SP600125 promoted ex vivo expansion of CD34+ cells. We also demonstrated that the activation of Lyn kinase and p110δ is a novel mechanism for Ech A to enhance ex vivo expansion of CD34+ cells. Ech A upregulated phospho-Src, phospho-Lyn, and p110δ expression. Furthermore, the Ech A-induced ex vivo expansion of CD34+ cells was inhibited by pretreatment with the Src family inhibitor PP1 and p110δ inhibitor CAL-101; PP1 blocked p110δ upregulation and PI3K/Akt activation, whereas CAL-101 and PI3K/Akt pathway inhibitor LY294002 did not block Src/Lyn activation. These results suggest that Ech A initially induces Src/Lyn activation, upregulates p110δ expression, and finally activates the PI3K/Akt pathway. CD34+ cells expanded in the presence of Ech A produced equal or more hematopoietic colony-forming cells than unexpanded CD34+ cells. In conclusion, Ech A promotes the ex vivo expansion of CD34+ cells through Src/Lyn-mediated p110δ expression, suppression of ROS generation, and p38-MAPK/JNK activation. Hence, Ech A is a potential candidate modality for the ex vivo, and possibly in vivo, expansion of CD34+ cells.


Assuntos
Antígenos CD34/metabolismo , Células Sanguíneas/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Naftoquinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo , Antracenos/farmacologia , Antioxidantes/metabolismo , Células Sanguíneas/metabolismo , Células Cultivadas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Piridinas/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
14.
Int J Nanomedicine ; 14: 5831-5848, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31534327

RESUMO

Purpose: In order to accelerate the tendon-bone healing processes and achieve the efficient osteointegration between the tendon graft and bone tunnel, we aim to design bioactive electrospun nanofiber membranes combined with tendon stem/progenitor cells (TSPCs) to promote osteogenic regeneration of the tendon and bone interface. Methods: In this study, nanofiber membranes of polycaprolactone (PCL), PCL/collagen I (COL-1) hybrid nanofiber membranes, poly(dopamine) (PDA)-coated PCL nanofiber membranes and PDA-coated PCL/COL-1 hybrid nanofiber membranes were successfully fabricated by electrospinning. The biochemical characteristics and nanofibrous morphology of the membranes, as well as the characterization of rat TSPCs, were defined in vitro. After co-culture with different types of electrospun nanofiber membranes in vitro, cell proliferation, viability, adhesion and osteogenic differentiation of TSPCs were evaluated at different time points. Results: Among all the membranes, the performance of the PCL/COL-1 (volume ratio: 2:1 v/v) group was superior in terms of its ability to support the adhesion, proliferation, and osteogenic differentiation of TSPCs. No benefit was found in this study to include PDA coating on cell adhesion, proliferation and osteogenic differentiation of TSPCs. Conclusion: The PCL/COL-1 hybrid electrospun nanofiber membranes are biocompatible, biomimetic, easily fabricated, and are capable of supporting cell adhesion, proliferation, and osteogenic differentiation of TSPCs. These bioactive electrospun nanofiber membranes may act as a suitable functional biomimetic scaffold in tendon-bone tissue engineering applications to enhance tendon-bone healing abilities.


Assuntos
Materiais Biocompatíveis/farmacologia , Osso e Ossos/fisiologia , Membranas Artificiais , Nanofibras/química , Células-Tronco/citologia , Tendões/citologia , Engenharia Tecidual/métodos , Animais , Osso e Ossos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Nanofibras/ultraestrutura , Osteogênese , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos
15.
Mater Sci Eng C Mater Biol Appl ; 104: 109955, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31500064

RESUMO

Calcium phosphate cement (CPC), functionalized with iron oxide nanoparticles (IONP), is of great promise to promote osteoinduction and new bone formation. In this work, the IONP powder was added into the CPC powder to fabricate CPC + IONP scaffolds and the effects of the novel composite on bone matrix formation and osteogenesis of human dental pulp stem cells (hDPSCs) were explored. A series of CPC + IONP magnetic scaffolds with different IONP contents (1%, 3% and 6%) were fabricated using 5% chitosan solution as the cement liquid. Western blotting and RT-PCR were used to analyze the signaling pathway. The IONP incorporation substantially enhanced the performance of CPC + IONP, with increases in both mechanical strength and cellular activities. The IONP addition greatly promoted the osteogenesis of hDPSCs, elevating the ALP activity, the expression of osteogenic marker genes and bone matrix formation with 1.5-2-fold increases. The 3% IONP incorporation showed the most enhancement among all groups. Activation of the extracellular signal-related kinases WNT/ß-catenin in DPSCs was observed, and this activation was attenuated by the WNT inhibitor DKK1. The results indicated that the osteogenic behavior of hDPSCs was likely driven by CPC + IONP via the WNT signaling pathway. In conclusion, incorporate IONP into CPC scaffold remarkably enhanced the spreading, osteogenic differentiation and bone mineral synthesis of stem cell. Therefore, this method had great potential for bone tissue engineering. The novel CPC + IONP composite scaffolds with stem cells are promising to provide an innovative strategy to enhance bone regenerative therapies.


Assuntos
Cimentos para Ossos/química , Cimentos para Ossos/farmacologia , Fosfatos de Cálcio/química , Compostos Férricos/química , Nanopartículas/química , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Quitosana/química , Cimentos Dentários/química , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Humanos , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte/química , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
16.
J Mater Sci Mater Med ; 30(9): 101, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31473826

RESUMO

Diabetes mellitus is the most common metabolic disorder with a high mortality and morbidity rate. A new promising strategy to treat DM is pancreatic tissue engineering. We described a 3D culture system accompanied by signaling factors to differentiate hEnSCs into IPCs in the presence of nZnO. We isolated EnSCs and cultured it in DMEM/F12 medium. Nanofibrous PLA/Cs scaffold was prepared through the electrospinning method. The morphological properties of the scaffolds and cells were evaluated by SEM. MTT assay was used to investigate the metabolic activity of the hEnSCs cultured on the scaffolds and a four-stage protocol was applied to differentiate hEnSCs. The differentiated cells were tested for pancreatic markers by immunocytochemistry, qRT-PCR and DTZ staining. The results of this study revealed that hEnSCs cultured on PLA/Cs scaffold and treated with nZnO can efficiently differentiate into IPCs. The examination of differentiated cell morphology showed their near similarity with pancreatic islet cells, and DTZ staining emphasized the presence of insulin granules inside their cytoplasm. Moreover, qRT-PCR and immunofluorescent staining results showed the efficient expression of specific gene markers of IPCs in resultant differentiated cells. Moreover, PLA/CS and nZnO were able to provide a good nanoenvironment for the differentiation of hEnSCs into IPCS the in presence of other molecules.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Endométrio/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Nanofibras/química , Células-Tronco/efeitos dos fármacos , Tecidos Suporte/química , Óxido de Zinco/farmacologia , Células Cultivadas , Feminino , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Teste de Materiais , Nanopartículas Metálicas/química , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Óxido de Zinco/química
17.
Molecules ; 24(18)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487916

RESUMO

Moringin [4-(α-L-rhamnosyloxy) benzyl isothiocyanate] is an isothiocyanate extracted from Moringa oleifera seeds. It is an antioxidant known for several biological properties useful in the treatment of neurodegenerative diseases. Several neurodegenerative disorders such as Parkinson's and Alzheimer's diseases are linked to dysfunctional mitochondria due to the resulting increase of Reactive Oxygen Species (ROS). Stem cell-based therapeutic treatments in neurodegenerative diseases provide an alternative strategy aimed to replace the impaired tissue. In this study were investigated the deregulated genes involved in mitophagy in the human periodontal ligament stem cells pretreated with moringin. The RNA-seq study reveals the downregulation of PINK1, with a fold change (FC) of -0.56, such as the genes involved in the phagophore formation (MAP1LC3B FC: -0.73, GABARAP FC: -0.52, GABARAPL1 FC: -0.70, GABARAPL2 FC: -0.39). The moringin pretreatment downregulates the pro-apoptotic gene BAX (-0.66) and upregulates the anti-apoptotic genes BCL2L12 (FC: 1.35) and MCL1 (FC: 0.36). The downregulation of the most of the caspases (CASP1 FC: -1.43, CASP4 FC: -0.18, CASP6 FC: -1.34, CASP7 FC: -0.46, CASP8 FC: -0.65) implies the inactivation of the apoptotic process. Our results suggest that mitochondrial dysfunctions induced by oxidative stress can be inhibited by moringin pretreatment in human periodontal ligament stem cells (hPDLSCs).


Assuntos
Expressão Gênica , Isotiocianatos/farmacologia , /genética , Ligamento Periodontal/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Biomarcadores , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Isotiocianatos/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Estrutura Molecular , Células-Tronco/citologia , Transcriptoma
18.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514329

RESUMO

Cartilage repair using tissue engineering is the most advanced clinical application in regenerative medicine, yet available solutions remain unsuccessful in reconstructing native cartilage in its proprietary form and function. Previous investigations have suggested that the combination of specific bioactive elements combined with a natural polymer could generate carrier matrices that enhance activities of seeded stem cells and possibly induce the desired matrix formation. The present study sought to clarify this by assessing whether a chitosan-hyaluronic-acid-based biomimetic matrix in conjunction with adipose-derived stem cells could support articular hyaline cartilage formation in relation to a standard chitosan-based construct. By assessing cellular development, matrix formation, and key gene/protein expressions during in vitro cultivation utilizing quantitative gene and immunofluorescent assays, results showed that chitosan with hyaluronic acid provides a suitable environment that supports stem cell differentiation towards cartilage matrix producing chondrocytes. However, on the molecular gene expression level, it has become apparent that, without combinations of morphogens, in the chondrogenic medium, hyaluronic acid with chitosan has a very limited capacity to stimulate and maintain stem cells in an articular chondrogenic state, suggesting that cocktails of various growth factors are one of the key features to regenerate articular cartilage, clinically.


Assuntos
Tecido Adiposo/citologia , Materiais Biomiméticos/farmacologia , Cartilagem Articular/fisiologia , Quitosana/farmacologia , Condrogênese , Ácido Hialurônico/farmacologia , Células-Tronco/citologia , Cartilagem Articular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura , Tecidos Suporte/química
19.
Artif Cells Nanomed Biotechnol ; 47(1): 3431-3437, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31411067

RESUMO

Presently, tissue engineering has been developed as an effective option in the restoration and repair of tissue defects. One of the tissue engineering strategies is to use both biodegradable scaffolds and stimulating factors for enhancing cell responses. In this study, the effect of zeolite was assessed on cell viability, proliferation, osteo/odontogenic differentiation, and mineralization of human dental pulp stem cells (hDPSCs) cultured on poly (ε-coprolactone) - poly (ethylene glycol)-poly (ε-caprolactone) (PCL-PEG-PCL) nanofibers. For this purpose, PCL-PEG-PCL nanofibrous scaffolds incorporated with zeolite were prepared via electrospinning. Both PCL-PEG-PCL and PCL-PEG-PCL/Zeolite nanofibrous scaffolds revealed bead-less constructions with average diameters of 430 nm and 437 nm, respectively. HDPSCs were transferred to PCL-PEG-PCL nanofibrous scaffolds containing zeolite nanoparticles. Cell adhesion and proliferation of hDPSCs and their osteo/odontogenic differentiation on these scaffolds were evaluated using MTT assay, Alizarin red S staining, and qRT-PCR assay. The results revealed that PCL-PEG-PCL/Zeolite nanofibrous scaffolds could support better cell adhesion, proliferation and osteogenic differentiation of hDPSCs and as such is expected to be a promising scaffold for bone tissue engineering applications.


Assuntos
Polpa Dentária/citologia , Nanofibras/química , Osteogênese/efeitos dos fármacos , Poliésteres/farmacologia , Polietilenoglicóis/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Zeolitas/química , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Poliésteres/química , Polietilenoglicóis/química , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte/química
20.
Exp Eye Res ; 187: 107767, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31437439

RESUMO

Limbal Stem Cell Deficiency (LSCD) is a painful and debilitating disease that results from damage or loss of the Corneal Epithelial Stem Cells (CESCs). Therapies have been developed to treat LSCD by utilizing epithelial stem cell transplants. However, effective repair and recovery depends on many factors, such as the source and concentration of donor stem cells, and the proper conditions to support these transplanted cells. We do not yet fully understand how CESCs heal wounds or how transplanted CESCs are able to restore transparency in LSCD patients. A major hurdle has been the lack of vertebrate models to study CESCs. Here we utilized a short treatment with Psoralen AMT (a DNA cross-linker), immediately followed by UV treatment (PUV treatment), to establish a novel frog model that recapitulates the characteristics of cornea stem cell deficiency, such as pigment cell invasion from the periphery, corneal opacity, and neovascularization. These PUV treated whole corneas do not regain transparency. Moreover, PUV treatment leads to appearance of the Tcf7l2 labeled subset of apical skin cells in the cornea region. PUV treatment also results in increased cell death, immediately following treatment, with pyknosis as a primary mechanism. Furthermore, we show that PUV treatment causes depletion of p63 expressing basal epithelial cells, and can stimulate mitosis in the remaining cells in the cornea region. To study the response of CESCs, we created localized PUV damage by focusing the UV radiation on one half of the cornea. These cases initially develop localized stem cell deficiency characteristics on the treated side. The localized PUV treatment is also capable of stimulating some mitosis in the untreated (control) half of those corneas. Unlike the whole treated corneas, the treated half is ultimately able to recover and corneal transparency is restored. Our study provides insight into the response of cornea cells following stem cell depletion, and establishes Xenopus as a suitable model for studying CESCs, stem cell deficiency, and other cornea diseases. This model will also be valuable for understanding the nature of transplanted CESCs, which will lead to progress in the development of therapeutics for LSCD.


Assuntos
Córnea/fisiologia , Doenças da Córnea/fisiopatologia , Epitélio Anterior/patologia , Células-Tronco/patologia , Cicatrização/fisiologia , Animais , Proliferação de Células , Reagentes para Ligações Cruzadas/toxicidade , Modelos Animais de Doenças , Epitélio Anterior/efeitos dos fármacos , Ficusina/toxicidade , Técnica Indireta de Fluorescência para Anticorpo , Homeostase/fisiologia , Marcação In Situ das Extremidades Cortadas , Fenótipo , Regeneração/fisiologia , Células-Tronco/efeitos dos fármacos , Raios Ultravioleta , Xenopus laevis
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