L-Ascorbic acid affect the DNA methytransferase expression in Mouse Embryonic Fibroblasts

Introduction: Induced pluripotent stem cells (iPSCs) can be generated from different source of cells with different efficiencies. Two DNA methyltransferases DNMT1 and DNMT3A have been shown to regulate epigenetically the gene expression involved in cell viability and reprogramming. L-ascorbic acid (L-AA) is a chemical factor that can accelerate reprogramming.  Here, we sought to investigate the effect of L-AA on DNMT1 and DNMT3A expressions. Materials and Methods: First, mouse embryonic fibroblasts at passage 3 were cultured in the presence of 10 µg/ml L-AA days for 5 days. Then, DNMT1 and DNMT3A expressions were determined using real-time PCR at days 3 and 5. Results: It was showed that L-AA could enhance DNMT-1 expression which involve in cell viability and decrease the DNMT3A which involve in cell differentiation. Conclusion: The results therefore suggest a new insight into L-AA mechanism impact on reprogramming process

Directed Pancreatic Acinar Differentiation of Mouse Embryonic Stem Cells via Embryonic Signalling Molecules and Exocrine Transcription Factors

Pluripotent embryonic stem cells (ESC) are a promising cellular system for generating an unlimited source of tissue for the treatment of chronic diseases and valuable in vitro differentiation models for drug testing. Our aim was to direct differentiation of mouse ESC into pancreatic acinar cells, which play key roles in pancreatitis and pancreatic cancer. To that end, ESC were first differentiated as embryoid bodies and sequentially incubated with activin A, inhibitors of Sonic hedgehog (Shh) and bone morphogenetic protein (BMP) pathways, fibroblast growth factors (FGF) and retinoic acid (RA) in order to achieve a stepwise increase in the expression of mRNA transcripts encoding for endodermal and pancreatic progenitor markers. Subsequent plating in Matrigel® and concomitant modulation of FGF, glucocorticoid, and folllistatin signalling pathways involved in exocrine differentiation resulted in a significant increase of mRNAs encoding secretory enzymes and in the number of cells co-expressing their protein products. Also, pancreatic endocrine marker expression was down-regulated and accompanied by a significant reduction in the number of hormone-expressing cells with a limited presence of hepatic marker expressing-cells. These findings suggest a selective activation of the acinar differentiation program. The newly differentiated cells were able to release α-amylase and this feature was greatly improved by lentiviral-mediated expression of Rbpjl and Ptf1a, two transcription factors involved in the maximal production of digestive enzymes. This study provides a novel method to produce functional pancreatic exocrine cells from ESC. © 2013 Delaspre et al.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII)-FEDER (PI052738 and PI080511 to A.S.; PI100094 and Tercel RD06/0010/ 0025 to B.S.). A.S. was supported by ISCIII and the Health Department of the Generalitat de Catalunya; F.D. was the recipient of a Graduate Fellowship from ISCIII and was also supported by Tercel; M.M. was the recipient of a Graduate Fellowship from the Generalitat de Catalunya.Peer Reviewe

Directed Differentiation of ES cells by pancreatic transcription factors p48, RBPJL and Mist1

A pesar de la abundancia de estudios realizados sobre el papel de las células acinares en las patologías exocrinas del páncreas (i.e. pancreatitis y cáncer), el estudio de las modificaciones producidas durante la diferenciación acinar en dichas patologías, se ha visto limitado por la escasez de modelos celulares no tumorales. Resultados previos de nuestro laboratorio, muestran que las células mES (células madre embrionarias de ratón )- pluripotentes y con la capacidad para generar tipos celulares especializados- pueden desarrollar un fenotipo acinar in vitro. Los objetivos de esta tesis han sido aumentar el contenido de enzimas digestivos así como las propiedades funcionales de las células generadas. Para ello se sobreexpresaron de forma estable p48, RBPJL y Mist1en células madre por transducción lentiviral de estos genes. Obtuvimos, gracias a una estrategia de infección en múltiples etapas, líneas celulares transgénicas mES que expresaban de forma constitutiva RBPJL y/o Mist1. La superimposición de la expresión de p48 por infección lentiviral en células en proceso de diferenciación dio lugar a una fuerte expresión de enzimas digestivos, con un patrón de regulación similar al que acontece in vivo durante el desarrollo pancreático. En esta inducción, tanto p48 como RPBJL son indispensables. Por otro lado, hemos mostrado un aumento elevado en la producción de varios componentes de la maquinaria secretota dependiente de Mist1. Además, hay que hacer notar ,que las células p48/RBPJL/Mist1 exhiben una regulada-secreción en respuesta a los secretagogos acinares y una mejor actividad de que la línea celular acinar 266-6. La expresión combinada de genes clave implicados en el desarrollo pancreático en células ES es un prometedor abordaje que nos llevará a una comprensión de los sutiles procesos del desarrollo exocrino pancreático.Despite known involvement of acinar cells in pancreatic exocrine pathologies (i.e pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. Our previous data showed that mES (murine embryonic stem) cells, which are pluripotent and have the ability to generate specialized cell types, can acquire an acinar phenotype in vitro. The aim of this work was to increase the digestive enzyme content of the generated cells as well as their functional properties based on stable overexpression of p48, RBPJL and Mist1 by lentiviral gene transduction. Thus, we engineered transgenic mES cell lines constitutively expressing RBPJL and/or Mist1 using a multi-step infection strategy. The superimposition of p48 expression by lentiviral infection of differentiating cells resulted in a strong expression of digestive enzymes, with a pattern of regulation similar to what occurs in vivo during pancreatic development. In this induction, both p48 and RPBJL are indispensable. On the other hand, we showed a high increase in the production of several components of the secretory machinery which was dependent of Mist1. Importantly, p48/RBPJL/Mist1 cells exhibited a regulated-secretory in response to acinar secretagogues and a better secretion activity than the 266-6 acinar cell line. Combined expression of key genes involved in pancreatic development in ES cells may be a promising approach to better understand subtle steps of pancreatic exocrine development

Directed Differentiation of ES cells by pancreatic transcription factors p48, RBPJL and Mist1

A pesar de la abundancia de estudios realizados sobre el papel de las células acinares en las patologías exocrinas del páncreas (i.e. pancreatitis y cáncer), el estudio de las modificaciones producidas durante la diferenciación acinar en dichas patologías, se ha visto limitado por la escasez de modelos celulares no tumorales. Resultados previos de nuestro laboratorio, muestran que las células mES (células madre embrionarias de ratón )- pluripotentes y con la capacidad para generar tipos celulares especializados- pueden desarrollar un fenotipo acinar in vitro. Los objetivos de esta tesis han sido aumentar el contenido de enzimas digestivos así como las propiedades funcionales de las células generadas. Para ello se sobreexpresaron de forma estable p48, RBPJL y Mist1en células madre por transducción lentiviral de estos genes. Obtuvimos, gracias a una estrategia de infección en múltiples etapas, líneas celulares transgénicas mES que expresaban de forma constitutiva RBPJL y/o Mist1. La superimposición de la expresión de p48 por infección lentiviral en células en proceso de diferenciación dio lugar a una fuerte expresión de enzimas digestivos, con un patrón de regulación similar al que acontece in vivo durante el desarrollo pancreático. En esta inducción, tanto p48 como RPBJL son indispensables. Por otro lado, hemos mostrado un aumento elevado en la producción de varios componentes de la maquinaria secretota dependiente de Mist1. Además, hay que hacer notar ,que las células p48/RBPJL/Mist1 exhiben una regulada-secreción en respuesta a los secretagogos acinares y una mejor actividad de que la línea celular acinar 266-6. La expresión combinada de genes clave implicados en el desarrollo pancreático en células ES es un prometedor abordaje que nos llevará a una comprensión de los sutiles procesos del desarrollo exocrino pancreático.Despite known involvement of acinar cells in pancreatic exocrine pathologies (i.e pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. Our previous data showed that mES (murine embryonic stem) cells, which are pluripotent and have the ability to generate specialized cell types, can acquire an acinar phenotype in vitro. The aim of this work was to increase the digestive enzyme content of the generated cells as well as their functional properties based on stable overexpression of p48, RBPJL and Mist1 by lentiviral gene transduction. Thus, we engineered transgenic mES cell lines constitutively expressing RBPJL and/or Mist1 using a multi-step infection strategy. The superimposition of p48 expression by lentiviral infection of differentiating cells resulted in a strong expression of digestive enzymes, with a pattern of regulation similar to what occurs in vivo during pancreatic development. In this induction, both p48 and RPBJL are indispensable. On the other hand, we showed a high increase in the production of several components of the secretory machinery which was dependent of Mist1. Importantly, p48/RBPJL/Mist1 cells exhibited a regulated-secretory in response to acinar secretagogues and a better secretion activity than the 266-6 acinar cell line. Combined expression of key genes involved in pancreatic development in ES cells may be a promising approach to better understand subtle steps of pancreatic exocrine development

Inductive effect of IDE1 on differentiation of human induced pluripotent stem cells into definitive endoderm cells by using PCL nanofibrous scaffold

Induced pluripotent cells have been considered as one of the most recent and best cell sources for the cell therapy. In this study, the differentiation potency of human iPS cells, cultured on scaffolds, which can differentiate into definitive endodermal cells as precursor for hepatocytes, pancreatic and lung cells, was studied. Embryoid bodies composed of pluripotent cells, were seeded on electrospinning nanofiber scaffold. The cells were differentiated into definitive endoderm using IDE1. Expression of definitive endoderm markers including Sox17, FoxA2 and GSC were confirmed by immunocytochemistry staining and qRT-PCR analysis. In the present study, morphology and viability of cells were evaluated by utilizing a scanning electron microscopy and MTT assay, respectively. The results demonstrated the positive effect of 3D cultures, using suitable factors, on definitive endoderm differentiation

Chemical Constituents and Antibacterial Activity of Essential Oil of Prangos ferulacea (L.) Lindl. Fruits: Essential oil of Prangos ferulacea

The essential oil of Prangos ferulacea (Apiaceae) fruits was obtained by hydrodistillation and analyzed by gas chromatography (GC) and GC-mass spectrometery (MS). Among the 39 identified constituents accounting for 99.99% of the total oil, the major components were chrysanthenyl acetate (26.53%), limonene (19.59%), alpha pinene (19.50%), delta-3-carene (6.56%), mesitaldehyde (6.09), and germacrene-B (3.55%). Antimicrobial activity of the essential oil was investigated against some gram positive and gram negative bacteria. The essential oil of P. ferulacea showed activity against Staphylococus aureus, S. epidermis, Eschrechia coli and Pseudomonas aeroginosa

A Review of the LARIAT Suture Delivery Device for Left Atrial Appendage Closure

The prevalence of atrial fibrillation (AF) is 1-2 % in the general population, and the risk of embolic stroke in AF patients is 4-5 times higher than that in the general population. AF-related strokes are often severe, and the rate of permanent disability is much higher among individuals who have AF-related strokes than in those who have strokes unrelated to AF. In patients with AF, more than 90 % of thrombi originate from the left atrial appendage (LAA). The purpose of this paper is to review the efficacy and safety of performing the LAA closure with the LARIAT Suture Delivery Device to prevent AF-related stroke in patients with contraindications to oral anticoagulant therapy

Directed Pancreatic Acinar Differentiation of Mouse Embryonic Stem Cells via Embryonic Signalling Molecules and Exocrine Transcription Factors

Pluripotent embryonic stem cells (ESC) are a promising cellular system for generating an unlimited source of tissue for the treatment of chronic diseases and valuable in vitro differentiation models for drug testing. Our aim was to direct differentiation of mouse ESC into pancreatic acinar cells, which play key roles in pancreatitis and pancreatic cancer. To that end, ESC were first differentiated as embryoid bodies and sequentially incubated with activin A, inhibitors of Sonic hedgehog (Shh) and bone morphogenetic protein (BMP) pathways, fibroblast growth factors (FGF) and retinoic acid (RA) in order to achieve a stepwise increase in the expression of mRNA transcripts encoding for endodermal and pancreatic progenitor markers. Subsequent plating in MatrigelH and concomitant modulation of FGF, glucocorticoid, and folllistatin signalling pathways involved in exocrine differentiation resulted in a significant increase of mRNAs encoding secretory enzymes and in the number of cells co-expressing their protein products. Also, pancreatic endocrine marker expression was down-regulated and accompanied by a significant reduction in the number of hormone-expressing cells with a limited presence of hepatic marker expressing-cells. These findings suggest a selective activation of the acinar differentiation program. The newly differentiated cells were able to release a-amylase and this feature was greatly improved by lentiviralmediated expression of Rbpjl and Ptf1a, two transcription factors involved in the maximal production of digestive enzymes

Human Bone Marrow Mesenchymal Stem Cell Behaviors on PCL/Gelatin Nanofibrous Scaffolds Modified with A Collagen IV-Derived RGD-Containing Peptide

Objective: We introduce an RGD (Arg-Gly-Asp)-containing peptide of collagen IV origin that possesses potent cell adhesion and proliferation properties. Materials and Methods: In this experimental study, the peptide was immobilized on an electrospun nanofibrous polycaprolactone/gelatin (PCL/Gel) hybrid scaffold by a chemical bonding approach to improve cell adhesion properties of the scaffold. An iodine-modified phenylalanine was introduced in the peptide to track the immobilization process. Native and modified scaffolds were characterized with scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). We studied the osteogenic and adipogenic differentiation potential of human bone marrow-derived mesenchymal stem cells (hBMSCs). In addition, cell adhesion and proliferation behaviors of hBMSCs on native and peptide modified scaffolds were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4',6-diamidino-2-phenylindole (DAPI) staining, and the results compared with tissue culture plate, as the control. Results: FTIR results showed that the peptide successfully immobilized on the scaffold. MTT assay and DAPI staining results indicated that peptide immobilization had a dramatic effect on cell adhesion and proliferation. Conclusion: This peptide modified nanofibrous scaffold can be a promising biomaterial for tissue engineering and regenerative medicine with the use of hBMSCs