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1.
Int J Biol Macromol ; 263(Pt 1): 130688, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38458294

RESUMO

This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation.


Assuntos
Queratinas , Peptídeo Hidrolases , Queratinas/metabolismo , Especificidade por Substrato , Peptídeo Hidrolases/metabolismo , Temperatura , Concentração de Íons de Hidrogênio
2.
J Agric Food Chem ; 71(50): 20062-20072, 2023 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-38078849

RESUMO

Reactive oxygen species (ROS) are crucial for signal transduction and the maintenance of cellular homeostasis. However, superfluous ROS may engender chronic pathologies. Feather keratin is a promising new source of antioxidant peptides that can eliminate excess ROS and potentially treat oxidative stress-related diseases, but the underlying mechanisms have remained elusive. This study investigated the antioxidant effects and mechanisms against H2O2-induced oxidative damage in HepG2 cells of the two latest discovered antioxidant peptides, CRPCGPTP (CP-8) and ANSCNEPCVR (AR-10), first decrypted from feather keratin. The results revealed that CP-8 and AR-10 did not exhibit cytotoxicity to HepG2 cells while reducing intracellular ROS accumulation. Simultaneously, they enhanced the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), thus alleviating H2O2-induced cell apoptosis. Molecular docking analysis demonstrated that CP-8, AR-10 interacted well with the key amino acids in the Kelch domain of Keap1, thereby directly disrupting the Keap1-Nrf2 interaction. The peptides' biosafety and antioxidant activity via Keap1/Nrf2 signaling lay the groundwork for further animal studies and applications as functional food additives.


Assuntos
Antioxidantes , Fator 2 Relacionado a NF-E2 , Animais , Humanos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Peróxido de Hidrogênio/toxicidade , Peróxido de Hidrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Queratinas , Plumas , Células Hep G2 , Simulação de Acoplamento Molecular , Estresse Oxidativo
3.
J Hematol Oncol ; 16(1): 62, 2023 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-37316891

RESUMO

BACKGROUND: The cytotoxicity of NK cells is largely dependent on IgG Fc receptor CD16a, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC). The high-affinity and non-cleavable CD16 (hnCD16) is developed and demonstrated a multi-tumor killing potential. However, the hnCD16 receptor activates a single CD16 signal and provides limited tumor suppression. How to exploit the properties of hnCD16 and incorporate NK cell-specific activation domains is a promising development direction to further improve the anti-tumor activity of NK cells. METHODS: To expand the applications of hnCD16-mediated ADCC for NK cell-based immunotherapy in cancer, we designed the hnCD16 Fusion Receptor (FR) constructs with the ectodomain of hnCD16 fused with NK cell-specific activating domains in the cytoplasm. FR constructs were transduced into CD16-negative NK cell line and human iPSC-derived NK (iNK) cells and effective FR constructs were screened. The up-regulation of immune activation- and cytokine-releasing-related pathways in FR-transduced NK cells was screened and validated by RNA sequencing and multiplex cytokines release assay, respectively. The tumor-killing efficiency was tested in vitro and in vivo via co-culture with tumor cell lines and xenograft mice-bearing human B-cell lymphoma, respectively. RESULTS: We screened the most effective combination to kill B cell lymphoma, which was fused with the ectodomain of hnCD16a, NK-specific co-stimulators (2B4 and DAP10) and CD3ζ in cytoplasmic domains. The screened construct showed excellent cytotoxicity effects and sharp multiple cytokines releasing both in the NK cell line and iNK cells. The transcriptomic analysis and validation assays of hnCD16- and hnCD16FR-transduced NK cells showed that hnCD16FR transduction remodeled immune-related transcriptome in NK cells, where significant upregulation of genes related to cytotoxicity, high cytokines releasing, induced tumor cell apoptosis, and ADCC in comparison with hnCD16 transduction were highlighted. In vivo xenograft studies demonstrated that a single low-dose regimen of engineered hnCD16FR iPSC-derived NK cells co-administered with anti-CD20 mAb treatment mediated potent activity and significantly improved survival. CONCLUSION: We developed a novel hnCD16FR construct that exhibits more potent cytotoxicity than reported hnCD16, which is a promising approach to treat malignancies with improved ADCC properties. We also offer a rationale for NK activation domains that remodel immune response to enhance CD16 signaling in NK cells.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Imunoterapia , Células Matadoras Naturais , Ativação Linfocitária , Linhagem Celular Tumoral , Citocinas
4.
Cell Mol Immunol ; 18(2): 427-439, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32939032

RESUMO

Vγ9Vδ2 T cells are promising candidates for cellular tumor immunotherapy. Due to their HLA-independent mode of action, allogeneic Vγ9Vδ2 T cells can be considered for clinical application. To apply allogeneic Vγ9Vδ2 T cells in adoptive immunotherapy, the methodology used to obtain adequate cell numbers with optimal effector function in vitro needs to be optimized, and clinical safety and efficacy also need to be proven. Therefore, we developed a novel formula to improve the expansion of peripheral γδ T cells from healthy donors. Then, we used a humanized mouse model to validate the therapeutic efficacy of expanded γδ T cells in vivo; furthermore, the expanded γδ T cells were adoptively transferred into late-stage liver and lung cancer patients. We found that the expanded cells possessed significantly improved immune effector functions, including proliferation, differentiation, and cancer cell killing, both in vitro and in the humanized mouse model. Furthermore, a phase I clinical trial in 132 late-stage cancer patients with a total of 414 cell infusions unequivocally validated the clinical safety of allogeneic Vγ9Vδ2 T cells. Among these 132 patients, 8 liver cancer patients and 10 lung cancer patients who received ≥5 cell infusions showed greatly prolonged survival, which preliminarily verified the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. Our clinical studies underscore the safety and efficacy of allogeneic Vγ9Vδ2 T-cell immunotherapy, which will inspire further clinical investigations and eventually benefit cancer patients.


Assuntos
Citotoxicidade Imunológica/imunologia , Imunoterapia Adotiva/métodos , Neoplasias Hepáticas/mortalidade , Neoplasias Pulmonares/mortalidade , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Células Alógenas , Animais , Proliferação de Células , Feminino , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Adulto Jovem
5.
FASEB J ; 34(11): 14336-14352, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32888237

RESUMO

Long noncoding RNAs (lncRNAs) are crucial in many cellular processes, yet relatively few have been shown to regulate human cardiomyocyte differentiation. Here, we demonstrate an essential role of GATA6 antisense RNA 1 (GATA6-AS1) in cardiomyocyte differentiation from human pluripotent stem cells (hPSCs). GATA6-AS1 is adjacent to cardiac transcription factor GATA6. We found that GATA6-AS1 was nuclear-localized and transiently upregulated along with GATA6 during the early stage of cardiomyocyte differentiation. The knockdown of GATA6-AS1 did not affect undifferentiated cell pluripotency but inhibited cardiomyocyte differentiation, as indicated by no or few beating cardiomyocytes and reduced expression of cardiomyocyte-specific proteins. Upon cardiac induction, the knockdown of GATA6-AS1 decreased GATA6 expression, altered Wnt-signaling gene expression, and reduced mesoderm development. Further characterization of the intergenic region between genomic regions of GATA6-AS1 and GATA6 indicated that the expression of GATA6-AS1 and GATA6 were regulated by a bidirectional promoter within the intergenic region. Consistently, GATA6-AS1 and GATA6 were co-expressed in several human tissues including the heart, similar to the mirror expression pattern of GATA6-AS1 and GATA6 during cardiomyocyte differentiation. Overall, these findings reveal a previously unrecognized and functional role of lncRNA GATA6-AS1 in controlling human cardiomyocyte differentiation.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , RNA Antissenso/genética , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Fator de Transcrição GATA6/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , RNA Antissenso/metabolismo , RNA Longo não Codificante/metabolismo
6.
Toxicol Sci ; 169(1): 280-292, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31059573

RESUMO

Alcohol use prior to and during pregnancy remains a significant societal problem and can lead to developmental fetal abnormalities including compromised myocardia function and increased risk for heart disease later in life. Alcohol-induced cardiac toxicity has traditionally been studied in animal-based models. These models have limitations due to physiological differences from human cardiomyocytes (CMs) and are also not suitable for high-throughput screening. We hypothesized that human-induced pluripotent stem cell-derived CMs (hiPSC-CMs) could serve as a useful tool to study alcohol-induced cardiac defects and/or toxicity. In this study, hiPSC-CMs were treated with ethanol at doses corresponding to the clinically relevant levels of alcohol intoxication. hiPSC-CMs exposed to ethanol showed a dose-dependent increase in cellular damage and decrease in cell viability, corresponding to increased production of reactive oxygen species. Furthermore, ethanol exposure also generated dose-dependent increased irregular Ca2+ transients and contractility in hiPSC-CMs. RNA-seq analysis showed significant alteration in genes belonging to the potassium voltage-gated channel family or solute carrier family, partially explaining the irregular Ca2+ transients and contractility in ethanol-treated hiPSC-CMs. RNA-seq also showed significant upregulation in the expression of genes associated with collagen and extracellular matrix modeling, and downregulation of genes involved in cardiovascular system development and actin filament-based process. These results suggest that hiPSC-CMs can be a novel and physiologically relevant system for the study of alcohol-induced cardiac toxicity.


Assuntos
Etanol/toxicidade , Cardiopatias/induzido quimicamente , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cardiotoxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Cardiopatias/metabolismo , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Medição de Risco
7.
J Immunother Cancer ; 7(1): 36, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30736852

RESUMO

BACKGROUND: Cholangiocarcinoma (CCA) is a highly aggressive and fatal tumor. CCA occurs in the epithelial cells of bile ducts. Due to increasing incidences, CCA accounts for 3% of all gastrointestinal malignancies. In addition to comprehensive treatments for cancer, such as surgery, chemotherapy, and radiotherapy, during the past few years, cellular immunotherapy has played an increasingly important role. As a result of our research, we have discovered the γδ T cell-based immunotherapy for CCA. CASE PRESENTATION: A 30-year-old male ( https://www.clinicaltrials.gov/ ID: NCT02425735) was diagnosed with recurrent mediastinal lymph node metastasis after liver transplantation because of Cholangiocarcinoma (stage IV). In the course of his therapy sessions, he only received allogenic γδ T cell immunotherapy from August, 2017 through February, 2018 (8 infusions in total). γδ T cells were expanded from peripheral blood mononuclear cells (PBMCs) of healthy donor, and ~ 4 × 108 cells were adoptive transferred to the patient. CONCLUSION: In the above case report of the Cholangiocarcinoma (stage IV) patient who had received liver transplantation and afterward was diagnosed with recurrent mediastinal lymph node metastasis, we clinically proved that allogenic γδ T cell treatment had no adverse effects. We observed that allogenic γδ T cell treatments positively regulated peripheral immune functions of the patient, depleted tumor activity, improved quality of life, and prolonged his life span. After 8 γδ T cell treatments, the size of lymph nodes was remarkably reduced with activity depletion. This clinical work suggested that allogenic γδ T cell immunotherapy could be developed into a promising therapy drug for CCA.


Assuntos
Neoplasias dos Ductos Biliares/terapia , Colangiocarcinoma/terapia , Imunoterapia , Linfócitos Intraepiteliais/transplante , Adulto , Neoplasias dos Ductos Biliares/imunologia , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/imunologia , Colangiocarcinoma/patologia , Humanos , Transplante de Fígado , Linfonodos/patologia , Metástase Linfática/patologia , Masculino
8.
Stem Cell Reports ; 9(2): 513-527, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28793247

RESUMO

Understanding molecules involved in differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes and endothelial cells is important in advancing hPSCs for cell therapy and drug testing. Here, we report that LGR5, a leucine-rich repeat-containing G-protein-coupled receptor, plays a critical role in hPSC differentiation into cardiomyocytes and endothelial cells. LGR5 expression was transiently upregulated during the early stage of cardiomyocyte differentiation, and knockdown of LGR5 resulted in reduced expression of cardiomyocyte-associated markers and poor cardiac differentiation. In contrast, knockdown of LGR5 promoted differentiation of endothelial-like cells with increased expression of endothelial cell markers and appropriate functional characteristics, including the ability to form tube-like structures and to take up acetylated low-density lipoproteins. Furthermore, knockdown of LGR5 significantly reduced the proliferation of differentiated cells and increased the nuclear translocation of ß-catenin and expression of Wnt signaling-related genes. Therefore, regulation of LGR5 may facilitate efficient generation of cardiomyocytes or endothelial cells from hPSCs.


Assuntos
Diferenciação Celular/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores Acoplados a Proteínas G/genética , Padronização Corporal/genética , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Mesoderma/citologia , Mesoderma/embriologia , Via de Sinalização Wnt
9.
Sci Rep ; 6: 30956, 2016 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-27492371

RESUMO

Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes.


Assuntos
Simulação por Computador , Mioblastos Cardíacos/fisiologia , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Técnicas de Cultura de Órgãos , Engenharia Tecidual , Simulação de Ausência de Peso
10.
Biomaterials ; 105: 66-76, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27509304

RESUMO

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 10(6) cells, a sensitivity (0.0001%) which was ∼2000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5(+) and TRA-1-60(+) cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications.


Assuntos
Rastreamento de Células/métodos , Células-Tronco Pluripotentes/química , Células-Tronco Pluripotentes/citologia , Análise Espectral Raman/métodos , Antígenos Embrionários Estágio-Específicos/análise , Ressonância de Plasmônio de Superfície/métodos , Biomarcadores/análise , Células Cultivadas , Humanos , Células-Tronco Pluripotentes/classificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
11.
Dis Model Mech ; 9(9): 927-39, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27491078

RESUMO

Although ß-blockers can be used to eliminate stress-induced ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), this treatment is unsuccessful in ∼25% of cases. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from these patients have potential for use in investigating the phenomenon, but it remains unknown whether they can recapitulate patient-specific drug responses to ß-blockers. This study assessed whether the inadequacy of ß-blocker therapy in an individual can be observed in vitro using patient-derived CPVT iPSC-CMs. An individual with CPVT harboring a novel mutation in the type 2 cardiac ryanodine receptor (RyR2) was identified whose persistent ventricular arrhythmias during ß-blockade with nadolol were abolished during flecainide treatment. iPSC-CMs generated from this patient and two control individuals expressed comparable levels of excitation-contraction genes, but assessment of the sarcoplasmic reticulum Ca(2+) leak and load relationship revealed intracellular Ca(2+) homeostasis was altered in the CPVT iPSC-CMs. ß-adrenergic stimulation potentiated spontaneous Ca(2+) waves and unduly frequent, large and prolonged Ca(2+) sparks in CPVT compared with control iPSC-CMs, validating the disease phenotype. Pursuant to the patient's in vivo responses, nadolol treatment during ß-adrenergic stimulation achieved negligible reduction of Ca(2+) wave frequency and failed to rescue Ca(2+) spark defects in CPVT iPSC-CMs. In contrast, flecainide reduced both frequency and amplitude of Ca(2+) waves and restored the frequency, width and duration of Ca(2+) sparks to baseline levels. By recapitulating the improved response of an individual with CPVT to flecainide compared with ß-blocker therapy in vitro, these data provide new evidence that iPSC-CMs can capture basic components of patient-specific drug responses.


Assuntos
Catecolaminas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Taquicardia Ventricular/tratamento farmacológico , Taquicardia Ventricular/patologia , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/fisiopatologia , Biomarcadores/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Flecainida/farmacologia , Flecainida/uso terapêutico , Homeostase/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Linhagem , Receptores Adrenérgicos beta/metabolismo , Taquicardia Ventricular/fisiopatologia
12.
Stem Cell Res ; 16(3): 740-50, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27131761

RESUMO

Enhancing the maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) will facilitate their applications in disease modeling and drug discovery. Previous studies suggest that cell alignment could enhance hPSC-CM maturation; however, the robustness of this approach has not been well investigated. To this end, we examined if the anisotropic orientation of hPSC-CMs imposed by the underlying aligned fibers within a 3D microenvironment could improve the maturation of hPSC-CMs. Enriched hPSC-CMs were cultured for two weeks on Matrigel-coated anisotropic (aligned) and isotropic (random) polycaprolactone (PCL) fibrous scaffolds, as well as tissue culture polystyrenes (TCPs) as a control. As expected, hPSC-CMs grown on the two types of fibrous scaffolds exhibited anisotropic and isotropic orientations, respectively. Similar to cells on TCPs, hPSC-CMs cultured on these scaffolds expressed CM-associated proteins and were pharmacologically responsive to adrenergic receptor agonists, a muscarinic agonist, and a gap junction uncoupler in a dose-dependent manner. Although hPSC-CMs grown on anisotropic fibrous scaffolds displayed the highest expression of genes encoding a number of sarcomere proteins, calcium handling proteins and ion channels, their calcium transient kinetics were slower than cells grown on TCPs. These results suggest that electrospun anisotropic fibrous scaffolds, as a single method, have limited effect on improving the maturation of hPSC-CMs.


Assuntos
Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Alicerces Teciduais/química , Cálcio/metabolismo , Cardiotônicos/farmacologia , Diferenciação Celular , Células Cultivadas , Humanos , Imuno-Histoquímica , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Poliésteres/química , Reação em Cadeia da Polimerase em Tempo Real , Imagem com Lapso de Tempo
13.
Biomaterials ; 50: 176-85, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25736507

RESUMO

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide a potential source of cells to repair injured ventricular myocardium. CM differentiation cultures contain non-cardiac cells and CMs of both nodal and working subtypes. Direct application of such cultures in clinical studies could induce arrhythmias; thus, further purification of working-type CMs from heterogeneous cultures is desirable. Here, we designed 10 molecular beacons (MBs) targeting NPPA mRNA, a marker associated with working-type CMs and highly up-regulated during differentiation. We examined these MBs by solution assays and established their specificity using NPPA-overexpressing CHO cells as well as hPSC-CMs. We selected one MB for subsequent CM subtype isolation using fluorescence-activated cell sorting because the signal-to-background ratio was the highest for this MB in solution assays and a linear correlation was observed between MB signals and the CM purity in differentiation cultures. Compared with cells with low MB signals, cells positively selected based on MB signal had higher expression levels of genes associated with working-type CMs and lower expression levels of genes associated with nodal-type CMs. Therefore, the MB-based method is capable of separating working-type CMs from nodal-type CMs with high specificity and throughput, potentially providing working-type CMs for biomedical applications.


Assuntos
Separação Celular/métodos , Sondas Moleculares/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Células CHO , Diferenciação Celular/genética , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Humanos , Camundongos , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
14.
Stem Cell Reports ; 3(2): 260-8, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25254340

RESUMO

Cardiomyocytes derived from human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, disease modeling, and drug discovery, all of which require enriched cardiomyocytes, ideally ones with mature phenotypes. However, current methods are typically performed in 2D environments that produce immature cardiomyocytes within heterogeneous populations. Here, we generated 3D aggregates of cardiomyocytes (cardiospheres) from 2D differentiation cultures of hPSCs using microscale technology and rotary orbital suspension culture. Nearly 100% of the cardiospheres showed spontaneous contractility and synchronous intracellular calcium transients. Strikingly, from starting heterogeneous populations containing ∼10%-40% cardiomyocytes, the cell population within the generated cardiospheres featured ∼80%-100% cardiomyocytes, corresponding to an enrichment factor of up to 7-fold. Furthermore, cardiomyocytes from cardiospheres exhibited enhanced structural maturation in comparison with those from a parallel 2D culture. Thus, generation of cardiospheres represents a simple and robust method for enrichment of cardiomyocytes in microtissues that have the potential use in regenerative medicine as well as other applications.


Assuntos
Albuminas/química , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Poliésteres/química , Actinina/metabolismo , Técnicas de Cultura Celular por Lotes , Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular , Fibroblastos/citologia , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Humanos , Contração Muscular , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fatores de Transcrição/metabolismo , Troponina I/metabolismo
15.
Neuropsychopharmacology ; 36(13): 2629-38, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21796102

RESUMO

Cigarette smoking is a social behavior. Smoking is also accompanied by distinctive gustatory and olfactory stimulation. However, none of these factors affecting nicotine intake are modeled in existing preclinical studies. We report a novel model of adolescent nicotine self-administration (SA) in rats where licking on drinking spouts was used as the operant behavior to activate the concurrent delivery of nicotine (i.v.) and an appetitive olfactogustatory (OG) cue, and social interaction was required for stable SA. The operant chamber was divided by a panel that separated the SA rat and another rat serving as the demonstrator, who had free access to the OG cue but did not receive nicotine. Orofacial contacts were permitted by the divider. Conditioned taste aversion prevented solo rats to self-administer nicotine. However, stable nicotine (15-30 µg/kg, free base) SA was established in the presence of demonstrator rats with free access to the OG cue. Omitting the olfactory component of the cue prevented the acquisition of nicotine SA. Mecamylamine, a nicotinic antagonist, reduced licking behavior. Familiar peers were more effective demonstrators in facilitating the acquisition of nicotine SA than were unfamiliar rats. No sex difference in nicotine intake was found. These data indicate that the contingent OG cue is associated with the aversive property of nicotine that prevents subsequent drug intake. Social information encoded in olfaction not only permits the establishment of stable nicotine SA but also enhances nicotine intake. These findings implicate adolescent social interactions in promoting smoking behavior by surmounting the aversive property of nicotine.


Assuntos
Nicotina/efeitos adversos , Olfato/fisiologia , Comportamento Social , Paladar/fisiologia , Tabagismo/fisiopatologia , Fatores Etários , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Feminino , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Autoadministração/psicologia , Olfato/efeitos dos fármacos , Fumar/fisiopatologia , Fumar/psicologia , Paladar/efeitos dos fármacos , Tabagismo/psicologia
16.
Phys Chem Chem Phys ; 12(36): 10996-1003, 2010 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-20668742

RESUMO

Highly ordered mesoporous ZrO(2)/carbon (FDU-15) composites have been synthesized via a facile evaporation induced triconstituent co-assembly (EISA) approach by using Pluronic F127 as a template and zirconium oxychloride octahydrate and resol as Zr and carbon sources. The synthesized mesoporous composites exhibit a highly ordered two-dimensional (2-D) hexagonal mesostructure with relatively high specific surface areas (up to 947 m(2) g(-1)), pore sizes around 3.8 nm and high pore volumes (up to 0.71 cm(3) g(-1)). The results clearly show that the crystalline zirconia nanoparticles (ca. 1.9-3.9 nm) are well-dispersed in amorphous matrices of the ordered mesoporous carbon FDU-15 materials, which construct the nanocomposites. The ordered mesostructures of the obtained ZrO(2)/FDU-15 composites can be well-retained even at the high pyrolysis temperature (up to 900 degrees C), suggesting a high thermal stability. The zirconia content of the ZrO(2)/FDU-15 composites can be tunable in a wide range (up to 47%). Moreover, the resultant mesoporous ZrO(2)/FDU-15 composites exhibit high catalytic activity in oxidative dehydrogenation (ODH) of ethylbenzene (EB) to styrene (ST), with high ethylbenzene conversion (59.6%) and styrene selectivity (90.4%), which is mainly attributed to the synergistic catalytic effect between the oxygen-containing groups located on the carbon pore walls and weakly basic sites of the nanocrystalline ZrO(2). Furthermore, the high specific surface areas and opening pore channels are also responsible for their high catalytic activity. Therefore, it is a very promising catalyst material in styrene production on an industrial scale.

17.
Langmuir ; 26(11): 8850-6, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20121245

RESUMO

Mesoporous silica encapsulating upconversion luminescence NaYF(4) nanorods with uniform core-shell structures have been successfully synthesized by the surfactant-assistant sol-gel process. The thickness of ordered mesoporous silica shells can be adjusted from 50 to 95 nm by varying the amount of hydrolyzed silicate oligomer precursors from tetraethyl orthosilicate (TEOS), which further influences the BET surface area, pore volume, and the luminescence intensity. After coated with mesoporous silica shells, the hydrophobic nanorods is rendered to hydropholic simultaneously. The obtained beta-NaYF(4)@SiO(2)@mSiO(2) core-shell nanorods possess high surface area (71.2-196 m(2) g(-1)), pore volume (0.07-0.17 cm(3) g(-1)), uniform pore size distribution (2.3 nm), and accessible channels. Furthermore, the uniform core-shell nanorods show strong upconversion luminescence property similar to the hexagonal upconversion cores. The open mesopores can not only provide convenient transmission channels but also offer the huge location for accommodation of large molecules, such as fluorescent dyes and quantum dots. The secondary-excitation fluorescence of Rhodamine B is generated from the upconversion rare-earth fluoride nanorods cores to the fluorescent dyes loaded in the mesoporous silica shells.

18.
J Colloid Interface Sci ; 342(2): 579-85, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19926096

RESUMO

Ordered nitrogen-containing mesoporous carbonaceous polymers have been synthesized via a direct triblock-copolymer-templating process by using soluble, low-molecular-weight urea-phenol-formaldehyde (UPF) resin as an organic precursor and amphiphilic triblock copolymer Pluronic F127 as a template. Characterization using small-angle X-ray scattering (SAXS), N(2) sorption, transmission electron microscopy (TEM), elemental analysis, thermogravimetric analysis (TG), Fourier transform infrared (FTIR), and water adsorption techniques reveals that the obtained nitrogen-containing mesoporous polymers possess ordered structures, high surface areas (385-420 m(2)/g), large pore sizes (3.1-3.6nm) and pore volumes (0.25-0.44cm(3)/g), and high nitrogen content (2.69-2.94%). Various mesostructures, such as two-dimensional (2-D) hexagonal (space group, p6mm) and 3-D body-centered cubic (Im3 m) symmetries, can be obtained by simply adjusting the mass ratio of UPF/F127. The content of nitrogen in the mesoporous polymers can also be easy varied by changing the amount of urea and the reaction time of UPF resin precursors. Compared with the nitrogen-free mesoporous polymer, the obtained mesoporous carbonaceous polymers show a more hydrophilic nature and thus evidently higher water adsorption capacity. The presence of nitrogen groups can also significantly improve the adsorption performance of Fe(III) ions.


Assuntos
Nitrogênio/química , Polímeros/química , Resinas Sintéticas/química , Adsorção , Cátions/química , Cátions/isolamento & purificação , Formaldeído/química , Ferro/química , Ferro/isolamento & purificação , Fenol/química , Porosidade , Ureia/química , Água/química
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