RESUMEN
Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44+ CD24-/low EpCAM+ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.
Asunto(s)
Tejido Adiposo/patología , Carcinogénesis/patología , Células Madre Neoplásicas/patología , Neoplasias de la Mama Triple Negativas/patología , Adipocitos/metabolismo , Adipocitos/patología , Adipogénesis/fisiología , Tejido Adiposo/metabolismo , Animales , Antígeno CD24/metabolismo , Carcinogénesis/metabolismo , Diferenciación Celular/fisiología , Fusión Celular/métodos , Línea Celular Tumoral , Movimiento Celular/fisiología , Condrogénesis/fisiología , Molécula de Adhesión Celular Epitelial/metabolismo , Femenino , Xenoinjertos/metabolismo , Xenoinjertos/patología , Humanos , Receptores de Hialuranos/metabolismo , Células MCF-7 , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Osteogénesis/fisiología , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are emerging tools for applications such as drug discovery and screening for pro-arrhythmogenicity and cardiotoxicity as leading causes for drug attrition. Understanding the electrophysiology (EP) of hPSC-CMs is essential but conventional manual patch-clamping is highly laborious and low-throughput. Here we adapted hPSC-CMs derived from two human embryonic stem cell (hESC) lines, HES2 and H7, for a 16-channel automated planar-recording approach for single-cell EP characterization. Automated current- and voltage-clamping, with an overall success rate of 55.0⯱â¯11.3%, indicated that 90% of hPSC-CMs displayed ventricular-like action potential (AP) and the ventricular cardiomyocytes (VCMs) derived from the two hESC lines expressed similar levels of INa, ICaL, Ikr and If and similarly lacked Ito and IK1. These well-characterized hPSC-VCMs could also be readily adapted for automated assays of pro-arrhythmic drug screening. As an example, we showed that flecainide (FLE) induced INa blockade, leftward steady-state inactivation shift, slowed recovery from inactivation in our hPSC-VCMs. Since single-cell EP assay is insufficient to predict drug-induced reentrant arrhythmias, hPSC-VCMs were further reassembled into 2D human ventricular cardiac monolayers (hvCMLs) for multi-cellular electrophysiological assessments. Indeed, FLE significantly slowed the conduction velocity while causing AP prolongation. Our RNA-seq data suggested that cell-cell interaction enhanced the maturity of hPSC-VCMs. Taken collectively, a combinatorial approach using single-cell EP and hvCMLs is needed to comprehensively assess drug-induced arrhythmogenicity.
Asunto(s)
Evaluación Preclínica de Medicamentos , Flecainida/efectos adversos , Ventrículos Cardíacos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Miocitos Cardíacos/efectos de los fármacos , Bloqueadores del Canal de Sodio Activado por Voltaje/efectos adversos , Canales de Sodio Activados por Voltaje/metabolismo , Potenciales de Acción/efectos de los fármacos , Automatización de Laboratorios , Diferenciación Celular , Línea Celular , Células Cultivadas , Fenómenos Electrofisiológicos/efectos de los fármacos , Estudios de Factibilidad , Sistema de Conducción Cardíaco/citología , Sistema de Conducción Cardíaco/efectos de los fármacos , Sistema de Conducción Cardíaco/metabolismo , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Reproducibilidad de los Resultados , Análisis de la Célula Individual , Canales de Sodio Activados por Voltaje/químicaRESUMEN
BACKGROUND: The mechanism of how reactive oxygen species (ROS) regulate cardiac differentiation in the long-run is unclear and the effect of pro-inflammatory cytokines secreted during myocardial infarction on the cardiac differentiation of embryonic stem cells (ESCs) is unknown. The aims of this study were 1) to investigate the effect of ROS on cardiac differentiation and the regulations of transcription factors in ESC differentiation cultures and 2) to investigate the effect of pro-inflammatory cytokines on the expression of cardiac structural genes and whether this effect is mediated through ROS signaling. METHODS: ESCs were differentiated using hanging drop method. Degree of cardiac differentiation was determined by the appearance of beating embryoid bodies (EBs) and by the expression of cardiac genes using real-time PCR and Western blot. Intracellular ROS level was examined by confocal imaging. RESULTS: H2O2-treated EBs were found to have enhanced cardiac differentiation in the long run as reflected by, firstly, an earlier appearance of beating EBs, and secondly, an upregulation in cardiac structural protein expression at both mRNA and protein levels. Also, ROS upregulated the expression of several cardiac-related transcription factors, and increased the post-translationally-activated transcription factors SRF and AP-1. IL-1ß, IL-10, IL-18 and TNF-α upregulated the expression of cardiac structural proteins and increased the ROS level in differentiating EBs. In addition, ROS scavenger reversed the cardiogenic effect of IL-10 and IL-18. CONCLUSIONS: These results demonstrated that ROS enhance cardiac differentiation of ESCs through upregulating the expression and activity of multiple cardiac-related transcription factors. IL-1ß, IL-10, IL-18 and TNF-α enhance cardiac differentiation and ROS may serve as the messenger in cardiogenic signaling from these cytokines.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Mediadores de Inflamación/metabolismo , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Citocinas/biosíntesis , Citocinas/metabolismo , Ratones , Infarto del Miocardio/patología , Factores de Transcripción/fisiologíaRESUMEN
Embryonic stem cells (ESCs) can self-renew indefinitely and differentiate into all cell lineages. Calcium is a universal second messenger which regulates a number of cellular pathways. Previous studies showed that store-operated calcium channels (SOCCs) but not voltage-operated calcium channels are present in mouse ESCs (mESCs). In this study, store-operated calcium entry (SOCE) was found to exist in mESCs using confocal microscopy. SOCC blockers lanthanum, 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365 reduced mESC proliferation in a concentration-dependent manner, suggesting that SOCE is important for ESC proliferation. Pluripotent markers, Sox-2, Klf-4, and Nanog, were down-regulated by 2-APB, suggesting that self-renewal property of mESCs relies on SOCE. 17ß-estradiol (E2) enhanced mESC proliferation. This enhanced proliferation was associated with an increment of SOCE. Both stimulated proliferation and increased SOCE could be reversed by SOCC blockers suggesting that E2 mediates its stimulatory effect on proliferation via enhancing SOCE. Also, cyclosporin A and INCA-6, inhibitors of calcineurin [phosphatase that de-phosphorylates and activates nuclear factor of activated T-cells (NFAT)], reversed the proliferative effect of E2, indicating that NFAT is involved in E2-stimulated proliferation. Interestingly, E2 caused the nuclear translocation of NFATc4, and this could be reversed by 2-APB. These results suggested that NFATc4 is the downstream target of E2-induced SOCE. The present investigation provides the first line of evidence that SOCE and NFAT are crucial for ESCs to maintain their unique characteristics. In addition, the present investigation also provides novel information on the mechanisms of how E2, an important female sex hormone, affects ESC proliferation.