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1.
Molecules ; 26(5)2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33800893

RESUMEN

In order to replace the huge amounts of copper salts used in citrus orchards, alternatives have been sought in the form of organic compounds of natural origin with activity against the causative agent of citrus canker, the phytopathogen Xanthomonas citri subsp. Citri. We synthesized a series of 4-alkoxy-1,2-benzene diols (alkyl-BDOs) using 1,2,4-benzenetriol (BTO) as a starting material through a three-step synthesis route and evaluated their suitability as antibacterial compounds. Our results show that alkyl ethers derived from 1,2,4-benzenetriol have bactericidal activity against X. citri, disrupting the bacterial cell membrane within 15 min. Alkyl-BDOs were also shown to remain active against the bacteria while in solution, and presented low toxicity to (human) MRC-5 cells. Therefore, we have demonstrated that 1,2,4-benzenetriol-a molecule that can be obtained from agricultural residues-is an adequate precursor for the synthesis of new compounds with activity against X. citri.


Asunto(s)
Antibacterianos/farmacología , Derivados del Benceno/farmacología , Citrus/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Hojas de la Planta/efectos de los fármacos , Xanthomonas/patogenicidad , Antibacterianos/química , Derivados del Benceno/química , Proliferación Celular , Citrus/microbiología , Fibroblastos/citología , Humanos , Hojas de la Planta/microbiología
2.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33807555

RESUMEN

Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.


Asunto(s)
Células Madre Embrionarias/citología , Organoides/citología , Animales , Biomarcadores/metabolismo , Blastocisto/citología , Blastocisto/metabolismo , Línea Celular , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Ratones , Ratones Endogámicos ICR , Sistema Nervioso/citología , Sistema Nervioso/metabolismo , Técnicas de Transferencia Nuclear , Organoides/metabolismo , Porcinos
3.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33809214

RESUMEN

Extracellular vesicles (EVs) are generated and secreted by cells into the circulatory system. Stem cell-derived EVs have a therapeutic effect similar to that of stem cells and are considered an alternative method for cell therapy. Accordingly, research on the characteristics of EVs is emerging. EVs were isolated from human epidural fat-derived mesenchymal stem cells (MSCs) and human fibroblast culture media by ultracentrifugation. The characterization of EVs involved the typical evaluation of cluster of differentiation (CD antigens) marker expression by fluorescence-activated cell sorting, size analysis with dynamic laser scattering, and morphology analysis with transmission electron microscopy. Lastly, the secreted levels of cytokines and chemokines in EVs were determined by a cytokine assay. The isolated EVs had a typical size of approximately 30-200 nm, and the surface proteins CD9 and CD81 were expressed on human epidural fat MSCs and human fibroblast cells. The secreted levels of cytokines and chemokines were compared between human epidural fat MSC-derived EVs and human fibroblast-derived EVs. Human epidural fat MSC-derived EVs showed anti-inflammatory effects and promoted macrophage polarization. In this study, we demonstrated for the first time that human epidural fat MSC-derived EVs exhibit inflammatory suppressive potency relative to human fibroblast-derived EVs, which may be useful for the treatment of inflammation-related diseases.


Asunto(s)
Diferenciación Celular/genética , Vesículas Extracelulares/genética , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Animales , Polaridad Celular/genética , Tratamiento Basado en Trasplante de Células y Tejidos , Quimiocinas/genética , Citocinas/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/trasplante , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Inflamación/genética , Inflamación/terapia , Macrófagos/metabolismo , Células Madre Mesenquimatosas/metabolismo
4.
Ann Agric Environ Med ; 28(1): 163-171, 2021 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-33775083

RESUMEN

INTRODUCTION: Currently, mobile phones and Wi-Fi are the most commonly used forms of telecommunication. The popularity of mobile telecommunications has made it necessary to investigate the problem more comprehensively and cautiously assess the possible risks, because never before in history has such a substantial proportion of the population been exposed to microwaves at comparably high levels. Some studies indicate that the high frequency electromagnetic radiation emitted by mobile phone and Wi-Fi connections can have a negative effect on human health, and can cause cancer. OBJECTIVE: The aim of the study was to investigate the influence of the radiofrquency electromagnetic field (RF-EMF) on the metaboloc activity and morphology of normal human cells (fibroblasts) and cancer cells (prostate cancer cells). MATERIAL AND METHODS: The cell cultures (human fibroblasts and prostate cancer cells) were exposed to RF-EMF at the frequency of 2.5 GHz for 24, 48 and 72h. To quantify changes in cell viability, the Cell Counting Kit - 8 was used. RESULTS: It was found that the RF electromagnetic field exposure caused a significant decrease in the viability of fibroblasts, and a significant increase in cancer cells. Morphological analysis did not show significant changes in both cell lines after exposure to RF-EMF. CONCLUSIONS: On the basis of the obtained results, the hypothesis can be formulated that a high frequency electromagnetic field can have harmful effects on human cells.


Asunto(s)
Línea Celular Tumoral/efectos de la radiación , Campos Electromagnéticos/efectos adversos , Fibroblastos/efectos de la radiación , Ondas de Radio/efectos adversos , Línea Celular , Teléfono Celular , Supervivencia Celular/efectos de la radiación , Exposición a Riesgos Ambientales/efectos adversos , Fibroblastos/citología , Humanos
5.
J Vis Exp ; (168)2021 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33720140

RESUMEN

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient's own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.


Asunto(s)
Fibroblastos/citología , Plasma Rico en Plaquetas/metabolismo , Actinas/metabolismo , Plaquetas/metabolismo , Técnicas de Cultivo de Célula , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Separación Celular , Células Cultivadas , Citoesqueleto/metabolismo , Humanos
6.
Methods Mol Biol ; 2265: 173-183, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704714

RESUMEN

Most currently available three-dimensional melanoma models have either focused on simplicity or were optimized for physiological relevance. Accordingly, these paradigms have been either composed of malignant cells only or they were sophisticated human skin equivalents featuring multiple cell types and skin-like organization. Here, an intermediate spheroid-based assay system is presented, which uses tri-cultures of human CCD-1137Sk fibroblasts, HaCaT keratinocytes, and SK-MEL-28 melanoma cells. Being made of cell lines, these spheroids can be reliably reproduced without any special equipment using standard culture procedures, and they feature different aspects of skin and early stage melanoma. Therefore, this kind of model can be useful for lead-compound testing or addressing fundamental principles of early melanoma formation.


Asunto(s)
Antineoplásicos/farmacología , Técnicas de Cocultivo/métodos , Fibroblastos/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Docetaxel/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología
7.
Int J Mol Sci ; 22(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652991

RESUMEN

A hydrogel system based on oxidized alginate covalently crosslinked with gelatin (ADA-GEL) has been utilized for different biofabrication approaches to design constructs, in which cell growth, proliferation and migration have been observed. However, cell-bioink interactions are not completely understood and the potential effects of free aldehyde groups on the living cells have not been investigated. In this study, alginate, ADA and ADA-GEL were characterized via FTIR and NMR, and their effect on cell viability was investigated. In the tested cell lines, there was a concentration-dependent effect of oxidation degree on cell viability, with the strongest cytotoxicity observed after 72 h of culture. Subsequently, primary human cells, namely fibroblasts and endothelial cells (ECs) were grown in ADA and ADA-GEL hydrogels to investigate the molecular effects of oxidized material. In ADA, an extremely strong ROS generation resulting in a rapid depletion of cellular thiols was observed in ECs, leading to rapid necrotic cell death. In contrast, less pronounced cytotoxic effects of ADA were noted on human fibroblasts. Human fibroblasts had higher cellular thiol content than primary ECs and entered apoptosis under strong oxidative stress. The presence of gelatin in the hydrogel improved the primary cell survival, likely by reducing the oxidative stress via binding to the CHO groups. Consequently, ADA-GEL was better tolerated than ADA alone. Fibroblasts were able to survive the oxidative stress in ADA-GEL and re-entered the proliferative phase. To the best of our knowledge, this is the first report that shows in detail the relationship between oxidative stress-induced intracellular processes and alginate di-aldehyde-based bioinks.


Asunto(s)
Alginatos/química , Materiales Biocompatibles/química , Células Endoteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Gelatina/química , Estrés Oxidativo/efectos de los fármacos , Alginatos/toxicidad , Animales , Materiales Biocompatibles/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/citología , Fibroblastos/citología , Gelatina/toxicidad , Humanos , Ratones , Células 3T3 NIH , Andamios del Tejido/química
8.
Methods Mol Biol ; 2269: 175-201, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687680

RESUMEN

Bench-to-bedside axis of therapeutic product development is currently being oriented towards minimum invasiveness on both ends-not only clinical application but harvesting of the starting biological material as well. This is particularly relevant for Advanced Therapy Medicinal Products and their specific legislative requirements, even more so in skin regeneration. It is precisely the skin equivalents and grafts that benefit from the minimum-to-noninvasive approach to a noteworthy extent, taking in account the sensitive nature of both skin harvesting and grafting.This chapter includes protocols for two separate steps of generating skin equivalent from the cells cultured from hair follicle outer root sheath. The first step is a non-pigmented epidermal equivalent generated from human keratinocytes from the outer root sheath named non-pigmented epidermal graft. The second step consists of co-cultivating human keratinocytes and human melanocytes from the outer root sheath, hereby producing a pigmented epidermal graft.


Asunto(s)
Dermis/metabolismo , Fibroblastos/metabolismo , Folículo Piloso/metabolismo , Queratinocitos/metabolismo , Melanocitos/metabolismo , Ingeniería de Tejidos , Técnicas de Cocultivo , Dermis/citología , Fibroblastos/citología , Folículo Piloso/citología , Humanos , Queratinocitos/citología , Melanocitos/citología
9.
Molecules ; 26(4)2021 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-33578815

RESUMEN

Coccoloba cowellii Britton (Polygonaceae) is an endemic and critically endangered plant that only grows in Camagüey, a province of Cuba. In this study, a total of 13 compounds were identified in a methanolic leaf extract, employing a dereplication of the UHPLC-HRMS data by means of feature-based molecular networking (FBMN) analysis in the Global Natural Products Social Molecular Network (GNPS), together with the interpretation of the MS/MS data and comparison with the literature. The major constituents were glucuronides and glycosides of myricetin and quercetin, as well as epichatechin-3-O-gallate, catechin, epicatechin and gallic acid, all of them being reported for the first time in C. cowellii leaves. The leaf extract was also tested against various microorganisms, and it showed a strong antifungal effect against Candida albicans ATCC B59630 (azole-resistant) (IC50 2.1 µg/mL) and Cryptococcus neoformans ATCC B66663 (IC50 4.1 µg/mL) with no cytotoxicity (CC50 > 64.0 µg/mL) on MRC-5 SV2 cells, determined by the resazurin assay. Additionally, the extract strongly inhibited COX-1 and COX-2 enzyme activity using a cell-free experiment in a dose-dependent manner, being significantly more active on COX-1 (IC50 4.9 µg/mL) than on COX-2 (IC50 10.4 µg/mL). The constituents identified as well as the pharmacological activities measured highlight the potential of C. cowellii leaves, increasing the interest in the implementation of conservation strategies for this species.


Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Fitoquímicos/farmacología , Extractos Vegetales/farmacología , Polygonaceae/química , Tripanocidas/farmacología , Bacterias/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Hongos/efectos de los fármacos , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Hojas de la Planta/química , Trypanosoma/efectos de los fármacos
10.
Methods Mol Biol ; 2273: 279-296, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604861

RESUMEN

In vitro epithelial models are valuable tools for both academic and industrial laboratories to investigate tissue physiology and disease. Epithelial tissues comprise the surface epithelium, basement membrane, and underlying supporting stromal cells. There are various types of epithelial tissue and they have a diverse and intricate architecture in vivo, which cannot be successfully recapitulated using two-dimensional (2D) cell culture. Tissue engineering strategies can be applied to bioengineer the organized, multilayered, and multicellular structure of epithelial tissues in vitro. Alvetex® is a porous, polystyrene scaffold that enables fibroblasts to synthesize a complex network of endogenous, humanized extracellular matrix proteins. This creates a physiologically relevant three-dimensional (3D) subepithelial microenvironment, enriched with mechanical and chemical cues, which supports the organization and differentiation of epithelial cells. Such technology has been used to bioengineer different epithelial architectures in vitro, including the simple, columnar structure of the intestine and the stratified, squamous, and keratinized structure of skin. Epithelial tissue models provide a useful platform for fundamental and translational research, with multifaceted applications including disease modeling, drug discovery, and product development.


Asunto(s)
Células Epiteliales/citología , Poliestirenos/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Células CACO-2 , Línea Celular , Fibroblastos/citología , Humanos , Queratinocitos/citología , Porosidad , Piel/citología
11.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33557232

RESUMEN

Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α4ß1 and α4ß7. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C' loop binding cleft within integrins α4ß1 and α4ß7. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C' loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α4ß1-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C' loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.


Asunto(s)
Diseño de Fármacos , Fibroblastos/efectos de los fármacos , Fibronectinas/metabolismo , Fibrosis/tratamiento farmacológico , Integrinas/metabolismo , Miofibroblastos/efectos de los fármacos , Péptidos/farmacología , Sitios de Unión , Diferenciación Celular , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fibronectinas/química , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Integrinas/química , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Simulación del Acoplamiento Molecular , Miofibroblastos/citología , Miofibroblastos/metabolismo , Unión Proteica , Dominios Proteicos , Transducción de Señal
12.
Mol Med Rep ; 23(4)2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33576455

RESUMEN

Rheumatoid arthritis (RA) is one of the most critical articular diseases, which is characterized by synovial hyperplasia and impaired quality of life. The clinical features of RA include chronic inflammation of the joints associated with synovial cell overgrowth. However, the mechanism regulating the outgrowth of fibroblast­like synoviocytes (FLS) is not fully understood. The present study reported that grap2 cyclin D interacting protein (GCIP), an inhibitor of DNA binding/differentiation (ID)­like helix­loop­helix protein, interacted with cAMP­response element­binding protein (CREB)­binding protein (CBP). Furthermore, GCIP repressed CREB­ and NF­κB­dependent gene expression by inhibiting CBP binding to RNA polymerase II complexes. GCIP depletion via small interfering RNA enhanced FLS growth, whereas stable GCIP expression suppressed the growth of 293 cells. In addition, GCIP depletion in FLS induced the expression of cyclin D1, a CREB target gene. The present study identified a novel inhibitory mechanism in which an ID protein may functionally target the transcriptional coactivator CBP. These results suggested that GCIP downregulation may be pivotal in FLS outgrowth.


Asunto(s)
Proteína de Unión a CREB/genética , Proliferación Celular/genética , Fibroblastos/metabolismo , Sinoviocitos/metabolismo , Factores de Transcripción/genética , Anciano , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Proteína de Unión a CREB/metabolismo , Movimiento Celular/genética , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Regulación hacia Abajo , Femenino , Fibroblastos/citología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Persona de Mediana Edad , Unión Proteica , Interferencia de ARN , Sinoviocitos/citología , Factores de Transcripción/metabolismo
13.
J Vis Exp ; (167)2021 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-33554972

RESUMEN

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 µM genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.


Asunto(s)
Bioingeniería/métodos , Corazón/fisiopatología , Esferoides Celulares/citología , Animales , Cardiotoxinas/farmacología , Recuento de Células , Separación Celular , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Colágeno/farmacología , Doxorrubicina/farmacología , Doxorrubicina/toxicidad , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Fibroblastos/citología , Geles , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Fijación del Tejido
14.
Methods Mol Biol ; 2244: 39-50, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33555581

RESUMEN

Primary human diploid fibroblasts are used routinely to study host/pathogen interactions of human cytomegalovirus (HCMV). Fibroblasts' ease of culture and tremendous permissiveness for infection allow the study of all facets of infection, an abbreviated list of which includes ligand-receptor interactions, activation of cell signaling responses, and dysregulation of the cell cycle and DNA repair processes. Another advantage to fibroblasts' permissiveness for HCMV is the capability to grow high titer stocks of virus in them. This chapter will discuss the production of viral stocks of HCMV in primary human fibroblasts, commencing with culturing and infection of cells and continuing through harvest, titration (determining the infectious capacity of a particular virus preparation), and storage of viral stocks for use in downstream experiments.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Citomegalovirus/genética , Fibroblastos/virología , Línea Celular , Células Cultivadas , Citomegalovirus/clasificación , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/virología , Diploidia , Fibroblastos/citología , Humanos , Modelos Biológicos , Replicación Viral
15.
Methods Mol Biol ; 2244: 19-38, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33555580

RESUMEN

Human cytomegalovirus is routinely isolated by inoculating fibroblast cultures with clinical specimens suspected of harboring HCMV and then monitoring the cultures for cytopathic effects characteristic of this virus. Initially, such clinical isolates are usually strictly cell-associated, but continued propagation in cell culture increases the capacity of an HCMV isolate to release cell-free infectious progeny. Once cell-free infection is possible, genetically homogenous virus strains can be purified by limiting dilution infections. HCMV strains can differ greatly with regard to the titers that can be achieved, the tropism for certain cell types, and the degree to which nonessential genes have been lost during propagation. As there is no ideal HCMV strain for all purposes, the choice of the most appropriate strain depends on the requirements of the particular experiment or project. In this chapter, we provide information that can serve as a basis for deciding which strain may be the most appropriate for a given experiment.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Citomegalovirus/genética , Tropismo Viral/genética , Citomegalovirus/clasificación , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/virología , Fibroblastos/citología , Humanos , Proyectos de Investigación , Tropismo Viral/fisiología , Replicación Viral
16.
Methods Mol Biol ; 2244: 115-132, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33555585

RESUMEN

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.


Asunto(s)
Citomegalovirus/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Cultivo Primario de Células/métodos , Línea Celular , Infecciones por Citomegalovirus/virología , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Humanos , ARN Interferente Pequeño/genética , Transfección/métodos , Proteínas Virales , Replicación Viral/fisiología
17.
Methods Mol Biol ; 2273: 151-158, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604851

RESUMEN

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial component of the placenta and supports the fetus during intrauterine life. Our understanding of these events is limited, particularly in human, because of ethical and legal restrictions and availability of adequate in vitro models would be very advantageous. Here we describe a method that converts human fibroblasts into trophoblast-like cells, combining the use of 5-azacytidine-CR (5-aza-CR) to erase the original cell phenotype and a cocktail containing bone morphogenetic protein 4 (BMP4) with inhibitors of the Activin/Nodal/ERK signaling pathways, to drive erased fibroblasts into the trophoblastic differentiation. This innovative method uses very easily accessible cells to derive trophoblast-like cells and it can be useful to study embryo implantation disorders related to aging.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Fibroblastos/citología , Trofoblastos/citología , Activinas/antagonistas & inhibidores , Animales , Azacitidina/farmacología , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Implantación del Embrión , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteína Nodal/antagonistas & inhibidores , Placenta/citología , Embarazo , Transducción de Señal , Piel/citología , Piel/crecimiento & desarrollo
18.
Nat Commun ; 12(1): 750, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33531466

RESUMEN

Muscle cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. However, molecular brakes restraining cell-cell fusion events have remained elusive. Here we show that transforming growth factor beta (TGFß) pathway is active in adult muscle cells throughout fusion. We find TGFß signaling reduces cell fusion, regardless of the cells' ability to move and establish cell-cell contacts. In contrast, inhibition of TGFß signaling enhances cell fusion and promotes branching between myotubes in mouse and human. Exogenous addition of TGFß protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFßR2 induces the formation of giant myofibers. Transcriptome analyses and functional assays reveal that TGFß controls the expression of actin-related genes to reduce cell spreading. TGFß signaling is therefore requisite to limit mammalian myoblast fusion, determining myonuclei numbers and myofiber size.


Asunto(s)
Músculo Esquelético/citología , Factor de Crecimiento Transformador beta/metabolismo , Adolescente , Adulto , Animales , Western Blotting , Fusión Celular , Células Cultivadas , Biología Computacional , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Regeneración/genética , Regeneración/fisiología , Células Madre/citología , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/genética , Adulto Joven
19.
Nat Commun ; 12(1): 517, 2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33483489

RESUMEN

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.


Asunto(s)
Fluorescencia , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Imagen Individual de Molécula/métodos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Microtúbulos/metabolismo , Fotometría/métodos
20.
Mol Cell ; 81(5): 1027-1042.e4, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33453166

RESUMEN

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.


Asunto(s)
Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Retroalimentación Fisiológica , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas Inhibidoras de STAT Activados/genética , ARN Helicasas/genética , Homeostasis del Telómero , Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Línea Celular , Línea Celular Tumoral , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/antagonistas & inhibidores , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Transducción de Señal , Sumoilación , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética
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