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1.
Angew Chem Int Ed Engl ; 60(40): 21824-21831, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34374184

RESUMO

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFß signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFß signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFß/SMAD signaling.

2.
Methods Mol Biol ; 2320: 65-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302648

RESUMO

In this chapter, we introduce the method for fabricating thick and anisotropic cardiac tissue for heart regeneration. Aligned and biodegradable nanofiber can be prepared by electrospinning Food and Drug Administration-approved poly (lactic-co-glycolic acid) on a rotating drum. After the nanofibers are transferred on to a polydimethylsiloxane frame, the cardiomyocytes could be plated on the nanofiber to form thick and anisotropic cardiac tissue rapidly. Cardiac tissue-like construct could be easily created by one-step method, and transplanted onto the hearts of myocardium infarction models and lead to their functional recovery.


Assuntos
Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Nanofibras/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Animais , Anisotropia , Células Cultivadas , Masculino , Miocárdio/citologia , Ratos , Ratos Nus , Engenharia Tecidual/métodos , Tecidos Suporte/química
3.
Commun Biol ; 3(1): 122, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170165

RESUMO

Directed differentiation methods allow acquisition of high-purity cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes has been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived cardiomyocytes can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, reentrant waves (ReWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with ReWs show higher maturation including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen-consumption rate, and enhanced contractile force. We subsequently use a mathematical model to interpret the origination, propagation, and long-term behavior of the ReWs within the SOTRs.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Cafeína/farmacologia , Cálcio/metabolismo , Células Cultivadas , Humanos , Mitocôndrias/metabolismo , Modelos Teóricos
4.
Adv Healthc Mater ; 8(13): e1900165, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31087474

RESUMO

Human pluripotent stem cells (hPSCs) have been widely used for various applications including disease modeling and regenerative medicine, among others. Recently, an increasing number of studies has focused on heterogeneity among hPSCs, which could affect cell quality and subsequent applications. In this study, a nanofibrous platform is developed for single human induced pluripotent stem cell isolation and culture. One type of single cell-derived subclone is established and found to have a distinct morphology compared to other subclones. When used for differentiation toward cardiomyocytes, this type of subclone demonstrates higher differentiation efficiency, increased maturation, and stronger beating compared to those derived from the other subclones. The findings provide a convenient method for single-cell isolation and culture, and demonstrate that variations in differentiation tendencies exist among subclones from the same cell line. This substrate adhesion-based selection process could be used to obtain cell lines with improved differentiation efficiency toward cardiomyocytes and other cell types, which would be advantageous for studies in various fields.


Assuntos
Diferenciação Celular , Miócitos Cardíacos/metabolismo , Nanofibras/química , Células-Tronco Pluripotentes/metabolismo , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Gelatina/química , Humanos , Cariótipo , Miócitos Cardíacos/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/citologia , Análise de Célula Única
5.
Stem Cell Reports ; 11(1): 142-156, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-30008324

RESUMO

We show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes' expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.


Assuntos
Junções Célula-Matriz/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Biomarcadores , Adesão Celular/genética , Diferenciação Celular , Proliferação de Células , Autorrenovação Celular/genética , Expressão Gênica , Humanos , Imunofenotipagem , Cariótipo , Modelos Biológicos
6.
Elife ; 62017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29231167

RESUMO

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is associated with congenital heart disease. However, little is known about how blood glucose impacts heart formation. Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic, electrophysiological, and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway. Blood glucose level in embryos is stable in utero during normal pregnancy, but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages. In a murine model of diabetic pregnancy, fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity. These data suggest that high glucose suppresses cardiac maturation, providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy.


Assuntos
Células-Tronco Embrionárias/citologia , Glucose/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Nucleotídeos/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Via de Pentose Fosfato , Gravidez , Edulcorantes/farmacologia
7.
Stem Cell Reports ; 9(5): 1546-1559, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29107590

RESUMO

High-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Transplante de Células-Tronco/métodos , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Masculino , Miócitos Cardíacos/transplante , Nanofibras/química , Poliglactina 910/química , Ratos , Ratos Nus , Tecidos Suporte/química
8.
Sci Rep ; 7(1): 8630, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819182

RESUMO

Pluripotent stem cell-derived cardiomyocytes show great promise in regenerating the heart after myocardial infarction; however, several uncertainties exist that must be addressed before clinical trials. One practical issue is graft survival following transplantation. Although a pro-survival cocktail with Matrigel has been shown to enhance graft survival, the use of Matrigel may not be clinically feasible. The purpose of this study was to test whether a hyaluronan-based hydrogel, HyStem, could be a substitute for Matrigel. Human induced pluripotent stem cell-derived cardiomyocytes diluted with HyStem alone, HyStem plus pro-survival factors, or a pro-survival cocktail with Matrigel (PSC/MG), were transplanted into a rat model of acute myocardial infarction. Histological analysis at 4 weeks post transplantation revealed that, among the three groups, recipients of PSC/MG showed the largest graft size. Additionally, the grafted cardiomyocytes in the recipients of PSC/MG had a more matured phenotype compared to those in the other two groups. These findings suggest that further studies will be required to enhance not only graft size, but also the maturation of grafted cardiomyocytes.


Assuntos
Matriz Extracelular/metabolismo , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Linhagem Celular , Transplante de Células/métodos , Modelos Animais de Doenças , Humanos , Hidrogéis/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Ratos Endogâmicos F344 , Ratos Nus
9.
Aging (Albany NY) ; 9(5): 1440-1452, 2017 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-28562313

RESUMO

We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Diferenciação Celular , Linhagem da Célula , Ataxia de Friedreich/metabolismo , Frequência Cardíaca , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Linhagem Celular , Separação Celular/métodos , Feminino , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Masculino , Miócitos Cardíacos/patologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
10.
Sci Rep ; 7: 43210, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28266620

RESUMO

Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals.


Assuntos
Diferenciação Celular , Fenômenos Eletrofisiológicos , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Técnicas de Cultura de Células , Humanos
11.
Angew Chem Int Ed Engl ; 56(7): 1765-1770, 2017 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-28067441

RESUMO

A major hurdle in stem cell therapy is the tumorigenic risk of residual undifferentiated stem cells. This report describes the design and evaluation of synthetic hybrid molecules that efficiently reduce the number of human induced pluripotent stem cells (hiPSCs) in cell mixtures. The design takes advantage of Kyoto probe 1 (KP-1), a fluorescent chemical probe for hiPSCs, and clinically used anticancer drugs. Among the KP-1-drug conjugates we synthesized, we found an exceptionally selective, chemically tractable molecule that induced the death of hiPSCs. Mechanistic analysis suggested that the high selectivity originates from the synergistic combination of transporter-mediated efflux and the cytotoxicity mode of action. The present study offers a chemical and mechanistic rationale for designing selective, safe, and simple reagents for the preparation of non-tumorigenic clinical samples.


Assuntos
Antineoplásicos/química , Separação Celular/métodos , Corantes Fluorescentes/química , Células-Tronco Pluripotentes Induzidas/citologia , Rodaminas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Corantes Fluorescentes/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Rodaminas/farmacologia
12.
Nature ; 538(7625): 388-391, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27723741

RESUMO

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.


Assuntos
Coração/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/transplante , Regeneração/fisiologia , Animais , Diferenciação Celular , Sobrevivência Celular , Feminino , Fibroblastos/citologia , Sobrevivência de Enxerto , Haplótipos , Imunossupressores , Macaca fascicularis , Complexo Principal de Histocompatibilidade/genética , Masculino , Contração Miocárdica/fisiologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/fisiopatologia , Fatores de Tempo , Transplante Homólogo
13.
Biofabrication ; 8(3): 035017, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27606680

RESUMO

Human induced pluripotent stem cells (hiPSCs) can be differentiated at high efficiency into cells of a targeting type but the resulting cell population has to be of high purity for clinical therapies to avoid teratomas. Herein, we report a microfluidic device with integrated and surface functionalised fishnet-like structures for specific cell capture. With the help of a flow derivation surface pattern, cells in solution are forced to cross the fishnet-like structure, resulting in high efficiency and selective retention of a chosen cell population. A suspension of hiPSCs and hiPSC-derived cardiomyocytes were used for device function validation. We found that a hiPSC capture rate over 80% can be achieved along with a remarkable increase in the CM population rate in the recovered suspension without affecting cell viability.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Linhagem Celular , Separação Celular , Sobrevivência Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Dispositivos Lab-On-A-Chip , Microscopia de Fluorescência , Miócitos Cardíacos/metabolismo , Propriedades de Superfície
14.
Stem Cells Int ; 2016: 2634013, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27446217

RESUMO

Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) hold high potential for use in drug assessment and myocardial regeneration. To create tissue-like constructs of CMs for extracellular monitoring, we placed aligned fibers (AFs) on the surface of a microelectrode array and then seeded hiPSC-CMs for subsequent monitoring for 14 days. As expected, the CMs organized into anisotropic and matured tissue and the extracellular recordings showed reduced premature beating higher signal amplitude and a higher probability of T-wave detection as compared to the culture without fibers. The CMs on the aligned fibers samples also exhibited anisotropic propagation of the field potential. These results therefore suggest that the hiPSC-CMs cultured on AFs can be used more reliably for cell based assays.

15.
Biochem Biophys Res Commun ; 469(3): 587-92, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26687948

RESUMO

Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-ß 42 (Aß42)/Aß40 and Aß43/Aß40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Células-Tronco Embrionárias Humanas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Presenilina-1/metabolismo , Animais , Diferenciação Celular , Modelos Animais de Doenças , Células-Tronco Embrionárias Humanas/patologia , Humanos , Mutação , Presenilina-1/genética , Regulação para Cima
16.
Respiration ; 88(3): 234-43, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25171691

RESUMO

BACKGROUND: There are few reports about sleep disturbances in patients with chronic obstructive pulmonary disease (COPD) in Asian countries. OBJECTIVES: To investigate the associations between sleep-disordered breathing (SDB) with hypoxemia and sleep quality, including sleep duration, in patients with COPD, we measured SDB and sleep quality including the objective sleep duration determined by an actigraph and portable monitoring. METHODS: A cross-sectional epidemiological health survey of 303 male employees (means ± SD: age 43.9 ± 8.2 years; BMI 24.0 ± 3.1) was conducted. Sleep quality was measured using the Epworth Sleepiness Scale (ESS) and the Pittsburgh Sleep Quality Index (PSQI). A respiratory disturbance index (RDI) ≥5 indicated SDB. RESULTS: Nineteen subjects (6.3%) had COPD. Among these, 11 (3.6%) had COPD with SDB (overlap syndrome). Sleep duration, ESS, and PSQI scores were not significantly different between COPD patients and normal control subjects. However, COPD patients had significantly longer sleep latency (p = 0.019), a lower sleep efficiency (p = 0.017), and a higher sleep fragmentation index (p = 0.041) and average activity (p = 0.0097) during sleep than control subjects. They also had a significantly higher RDI and more severe desaturation during sleep than control subjects (p < 0.01). The differences remained after adjustment for age and BMI but disappeared following adjustment for RDI. CONCLUSIONS: COPD patients with even mild-to-moderate airflow limitations had nocturnal desaturation and RDI-related impaired sleep quality without significant symptoms.


Assuntos
Emprego/estatística & dados numéricos , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Apneia Obstrutiva do Sono/epidemiologia , População Urbana/estatística & dados numéricos , Actigrafia , Adulto , Estudos Transversais , Humanos , Hipóxia/epidemiologia , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Síndromes da Apneia do Sono/epidemiologia , Privação do Sono/epidemiologia , Fatores de Tempo
17.
Stem Cell Reports ; 2(5): 734-45, 2014 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-24936458

RESUMO

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production.


Assuntos
Células-Tronco Pluripotentes/citologia , Polímeros/química , Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Cariotipagem , Microscopia Eletrônica de Transmissão , Células-Tronco Pluripotentes/metabolismo , Teratoma/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Cell Rep ; 6(6): 1165-1174, 2014 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-24613351

RESUMO

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.


Assuntos
Corantes Fluorescentes/química , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/citologia , Sondas Moleculares/química , Células-Tronco Pluripotentes/química , Células-Tronco Pluripotentes/citologia , Animais , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência/métodos
20.
Artigo em Inglês | MEDLINE | ID: mdl-23318682

RESUMO

Accumulating evidence indicates that ERK MAP kinase signaling plays an important role in the regulation of the circadian clock, especially in the clock-resetting mechanism in the suprachiasmatic nucleus (SCN) in mammals. Previous studies have also shown that ERK phosphorylation exhibits diurnal variation in the SCN. However, little is known about circadian regulation of ERK signaling in peripheral tissues. Here we show that the activity of Ras/ERK signaling exhibits circadian rhythms in mouse liver. We demonstrate that Ras activation, MEK phosphorylation, and ERK phosphorylation oscillate in a circadian manner. As the oscillation of ERK phosphorylation is lost in Cry1/Cry2 double-knockout mice, Ras/ERK signaling should be under the control of the circadian clock. Furthermore, expression of MAP kinase phosphatase-1 (Mkp-1) shows diurnal changes in liver. These results indicate that Ras/ERK signaling is strictly regulated by the circadian clock in liver, and suggest that the circadian oscillation of the activities of Ras, MEK, and ERK may regulate diurnal variation of liver function and/or homeostasis.(Communicated by Shigekazu NAGATA, M.J.A.).


Assuntos
Relógios Circadianos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fígado/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Fígado/enzimologia , Camundongos , Fosforilação
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