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Halting breast cancer metastatic relapses following primary tumor removal and the clinical dormant phase, remains challenging, due to a lack of specific vulnerabilities to target during dormancy. To address this, we conducted genome-wide CRISPR screens on two breast cancer cell lines with distinct dormancy properties: 4T1 (short-term dormancy) and 4T07 (prolonged dormancy). We discovered that loss of class-III PI3K, Pik3c3, revealed a unique vulnerability in 4T07 cells. Surprisingly, dormancy-prone 4T07 cells exhibited higher mTORC1 activity than 4T1 cells, due to lysosome-dependent signaling occurring at the cell periphery. Pharmacological inhibition of Pik3c3 counteracted this phenotype in 4T07 cells, and selectively reduced metastasis burden only in the 4T07 dormancy-prone model. This mechanism was also detected in human breast cancer cell lines in addition to a breast cancer patient-derived xenograft supporting that it may be relevant in humans. Our findings suggest dormant cancer cell-initiated metastasis may be prevented in patients carrying tumor cells that display PIK3C3-peripheral lysosomal signaling to mTORC1. Statement of Significance: We reveal that dormancy-prone breast cancer cells depend on the class III PI3K to mediate a constant peripheral lysosomal positioning and mTORC1 hyperactivity. Targeting this pathway might blunt breast cancer metastasis.
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The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified â¼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.
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Factores de Ribosilacion-ADP , Fosfolipasa D , Transducción de Señal , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Humanos , Fosfolipasa D/metabolismo , Fosfolipasa D/genética , Células HEK293 , Animales , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Mapeo de Interacción de ProteínasRESUMEN
The ADP-ribosylation factors (ARFs) and ARF-like (ARLs) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we utilized proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ~3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.
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Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293 cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.
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Vía de Señalización Hippo , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas , Ribonucleasa P , Humanos , Células HeLa , Vía de Señalización Hippo/fisiología , Ribonucleasa P/metabolismo , Células HEK293 , Subunidades de Proteína/metabolismoRESUMEN
Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1-/- mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin-null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.
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Mioblastos , Transducción de Señal , Ratones , Animales , Mioblastos/metabolismo , Ratones Noqueados , Músculos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismoRESUMEN
The tight coordination of diverse cytoskeleton elements is required to support several dynamic cellular processes involved in development and tissue homeostasis. The spectraplakin-family of proteins are composed of multiple domains that provide versatility to connect different components of the cytoskeleton, including the actin microfilaments, microtubules and intermediates filaments. Spectraplakins act as orchestrators of precise cytoskeletal dynamic events. In this review, we focus on the prototypical spectraplakin MACF1, a protein scaffold of more than 700 kDa that coordinates the crosstalk between actin microfilaments and microtubules to support cell-cell connections, cell polarity, vesicular transport, proliferation, and cell migration. We will review over two decades of research aimed at understanding the molecular, physiological and pathological roles of MACF1, with a focus on its roles in developmental and cancer. A deeper understanding of MACF1 is currently limited by technical challenges associated to the study of such a large protein and we discuss ideas to advance the field.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.
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Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/metabolismo , Secuencia de Aminoácidos/fisiología , Animales , Drosophila , Humanos , Unión Proteica/fisiología , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Keratin intermediate filaments (IFs) are the major cytoskeletal component in epithelial cells. The dynamics of keratin IFs have been described to depend mostly on the actin cytoskeleton, but the rapid transport of fully polymerized keratin filaments has not been reported. In this work, we used a combination of photoconversion experiments and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 genome editing to study the role of microtubules and microtubule motors in keratin filament transport. We found that long keratin filaments, like other types of IFs, are transported along microtubules by kinesin-1. Our data revealed that keratin and vimentin are nonconventional kinesin-1 cargoes because their transport did not require kinesin light chains, which are a typical adapter for kinesin-dependent cargo transport. Furthermore, we found that the same domain of the kinesin heavy chain tail is involved in keratin and vimentin IF transport, strongly suggesting that multiple types of IFs move along microtubules using an identical mechanism.-Robert, A., Tian, P., Adam, S. A., Kittisopikul, M., Jaqaman, K., Goldman, R. D., Gelfand, V. I. Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport.
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Filamentos Intermedios/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Cinesinas/fisiología , Microtúbulos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Vimentina/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Cinesinas/antagonistas & inhibidores , Ratones , Ratones Noqueados , Microscopía FluorescenteRESUMEN
The type III intermediate filament protein vimentin was once thought to function mainly as a static structural protein in the cytoskeleton of cells of mesenchymal origin. Now, however, vimentin is known to form a dynamic, flexible network that plays an important role in a number of signaling pathways. Here, we describe various methods that have been developed to investigate the cellular functions of the vimentin protein and intermediate filament network, including chemical disruption, photoactivation and photoconversion, biolayer interferometry, soluble bead binding assay, three-dimensional substrate experiments, collagen gel contraction, optical-tweezer active microrheology, and force spectrum microscopy. Using these techniques, the contributions of vimentin to essential cellular processes can be probed in ever further detail.
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Filamentos Intermedios/metabolismo , Vimentina/metabolismo , Animales , Colágeno/metabolismo , HumanosRESUMEN
The mechanical properties of vertebrate cells are largely defined by the system of intermediate filaments (IF). As part of a dense network, IF polymers are constantly rearranged and relocalized in the cell to fulfill their duty as cells change shape, migrate, or divide. With the development of new imaging technologies, such as photoconvertible proteins and super-resolution microscopy, a new appreciation for the complexity of IF dynamics has emerged. This review highlights new findings about the transport of IF, the remodeling of filaments by a process of severing and re-annealing, and the subunit exchange that occurs between filament precursors and a soluble pool of IF. We will also discuss the unique dynamic features of the keratin IF network. Finally, we will speculate about how the dynamic properties of IF are related to their functions.
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Filamentos Intermedios/ultraestructura , Animales , Proteínas del Citoesqueleto/fisiología , Proteínas del Citoesqueleto/ultraestructura , Humanos , Multimerización de ProteínaRESUMEN
BACKGROUND: It has been suggested that public awareness of aphasia is vital for extending services, research support, social inclusion and targeted raising of awareness. Earlier studies show that knowledge of aphasia varies across a range of variables, but is very low compared with other conditions. AIMS: To report a series of surveys of public awareness of aphasia from six countries, the largest study conducted this far. METHODS & PROCEDURES: Surveys were conducted in Argentina (N = 800), Canada (N = 831), Croatia (N = 400), Greece (N = 800), Norway (N = 251) and Slovenia (N = 400) using the same methodology requesting information on age, sex and occupation, asking whether respondents had heard of aphasia and where they had heard of it. Respondents were tested on their levels of knowledge of aphasia. OUTCOMES & RESULTS: Results revealed low levels of awareness of aphasia in countries surveyed with marked variability that appeared to interact with occupation, country, age and sex. We surveyed 3483 respondents (mean age = 43.16; SD = 17.68). Between 60% (Croatia) and 16% (Slovenia) said they had heard of aphasia (37.1% overall), but those with actual knowledge ranged from 13.9% (Norway) to 1.0% (Argentina). The combined mean of those with basic knowledge was 9.2%. Those who had heard of aphasia were younger; and females had higher levels of awareness. We also found associations between socio-economic status and awareness. Those working in health, social and educational spheres had the highest levels. Respondents mainly heard about aphasia through the media and work or personal contact with aphasia. CONCLUSIONS & IMPLICATIONS: Levels of awareness are low everywhere in absolute terms, and relative to the awareness of other conditions, with significant variability between countries, sex and socio-economic status. We examine how surveys can be utilized to plan ways to increase understanding and discuss the comparison of awareness of aphasia with other conditions.
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Afasia/diagnóstico , Afasia/psicología , Concienciación , Comparación Transcultural , Opinión Pública , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Educación en Salud , Humanos , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Intermediate filaments (IFs) are a component of the cytoskeleton capable of profound reorganization in response to specific physiological situations, such as differentiation, cell division, and motility. Various mechanisms were proposed to be responsible for this plasticity depending on the type of IF polymer and the biological context. For example, recent studies suggest that mature vimentin IFs (VIFs) undergo rearrangement by severing and reannealing, but direct subunit exchange within the filament plays little role in filament dynamics at steady state. Here, we studied the dynamics of subunit exchange in VIF precursors, called unit-length filaments (ULFs), formed by the lateral association of eight vimentin tetramers. To block vimentin assembly at the ULF stage, we used the Y117L vimentin mutant (vimentin(Y117L)). By tagging vimentin(Y117L) with a photoconvertible protein mEos3.2 and photoconverting ULFs in a limited area of the cytoplasm, we found that ULFs, unlike mature filaments, were highly dynamic. Subunit exchange among ULFs occurred within seconds and was limited by the diffusion of soluble subunits in the cytoplasm rather than by the association and dissociation of subunits from ULFs. Our data demonstrate that cells expressing vimentin(Y117L) contained a large pool of soluble vimentin tetramers that was in rapid equilibrium with ULFs. Furthermore, vimentin exchange in ULFs required ATP, and ATP depletion caused a dramatic reduction of the soluble tetramer pool. We believe that the dynamic exchange of subunits plays a role in the regulation of ULF assembly and the maintenance of a soluble vimentin pool during the reorganization of filament networks.
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Adenosina Trifosfato/metabolismo , Filamentos Intermedios/metabolismo , Precursores de Proteínas/metabolismo , Vimentina/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Humanos , Filamentos Intermedios/química , Filamentos Intermedios/genética , Cinética , Ratones Noqueados , Microscopía Confocal , Modelos Biológicos , Mutación Missense , Multimerización de Proteína , Precursores de Proteínas/química , Precursores de Proteínas/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Imagen de Lapso de Tiempo/métodos , Vimentina/química , Vimentina/genéticaRESUMEN
PURPOSE: Despite the relatively high prevalence of aphasia, research indicates that, world-wide, public awareness of aphasia is lacking. Of the surveys that have been conducted internationally, none has studied the Canadian public's awareness of aphasia. The purpose of the present survey was to assess public awareness and basic knowledge of aphasia of individuals in southern Ontario, Canada. METHOD: Using the same questionnaire that has been used in other countries, face-to-face surveys were conducted in public places (e.g. parks) at various locations in southern Ontario. Respondents were asked questions pertaining to their awareness and knowledge of aphasia. The number of surveys retained for analysis was 831. In addition to an evaluation of public awareness and knowledge of aphasia, the potential influences of age, gender, and occupation were analysed. For those who had heard of aphasia, questions were asked to determine how or where they had heard of aphasia. RESULT: Consistent with the literature, overall public awareness and basic knowledge of aphasia in southern Ontario was found to be limited. The factors of age, gender and occupation were found to influence the results. CONCLUSION: This investigation supports the need for better promotion of aphasia awareness to the public in southern Ontario and, by extension, in Canada.
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Afasia , Conocimientos, Actitudes y Práctica en Salud , Adolescente , Adulto , Anciano , Femenino , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Ontario , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.
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Actinas/metabolismo , Filamentos Intermedios/metabolismo , Microtúbulos/metabolismo , Vimentina/metabolismo , Quinasas p21 Activadas/metabolismo , Quinasas Asociadas a rho/metabolismo , Adenosina Trifosfato/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Dineínas/metabolismo , Células HeLa , Humanos , Filamentos Intermedios/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Tiazolidinas/farmacologíaRESUMEN
Environmental conditions in DU encourage biofilm development. This biofilm may represent a risk for patients and dental staff exposed to water and aerosols generated during dental cares, particularly for immunocompromised persons. A survey was conducted on the 175 dental surgeons of the department of Vienne (France) to investigate the motivations of dental practitioners to renew their DU, their awareness levels with respect to infectious risks related to water circulating within DU, and methods used for the maintenance of DU waterlines. These dentists were only partially aware of the need for maintaining DU waterlines. For this maintaining, chemical treatments and purges of pipes were carried out by 88% and 91.5% of dentists respectively ; chemical treatments were usually on a continous mode and dentists seemed to have complete confidence in their DU supplier regarding the choice and the use of chemical treatments. Flushes were performed only once per day in most cases (63%). This survey also highlighted that dentists were not enough aware of water related infectous risk, even though 68% estimated that the development of a biofilm within DU waterlines was an actual risk. Finally, very positively, dentists strongly indicated their wish to be more informed regarding all these risks. Although these results are based on a relatively small sample, corresponding to dentists of a French department, they clearly suggest that awareness of dental surgeons is still insufficient and must be performed to permit an effective prevention of infectious risk related to DU waterlines.
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Actitud del Personal de Salud , Equipo Dental/microbiología , Odontólogos/psicología , Control de Infección Dental/métodos , Microbiología del Agua , Biopelículas , Infección Hospitalaria/prevención & control , Desinfectantes Dentales/uso terapéutico , Educación Continua en Odontología , Contaminación de Equipos/prevención & control , Diseño de Equipo , Humanos , Mantenimiento , Motivación , Purificación del Agua/instrumentación , Purificación del Agua/métodosRESUMEN
Megakaryocytes (MK) are hematopoietic cells present in the bone marrow that are responsible for the production and release of platelets in the circulation. Given their very low frequency (<1%), human MK often need to be derived in culture to study their development or to generate sufficient material for biological studies. This chapter describes a simplified 14-day culture protocol that efficiently leads to the production of MK and platelets from cord blood enriched progenitor cells. A serum-free medium is suggested for the growth of the CB cells together with an optimized cytokine cocktail developed specifically for MK differentiation, expansion, and maturation. Methodologies for flow cytometry analysis, MK and platelets estimation, and MK progenitor assay are also presented.
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Plaquetas/citología , Diferenciación Celular , Ensayo de Unidades Formadoras de Colonias/métodos , Sangre Fetal/citología , Megacariocitos/citología , Células Madre/citología , Antígenos CD34/metabolismo , Técnicas de Cultivo de Célula , Separación Celular , Criopreservación , Citometría de Flujo , Humanos , Megacariocitos/metabolismo , Ploidias , Células Madre/metabolismoRESUMEN
Modern dental chair units consist of a network of interconnected narrow-bore plastic tubes called dental unit waterlines (DUWLs). The water delivered by these DUWLs acts as both a coolant for a range of instruments and an irrigant during dental treatments. The quality of water is of considerable importance because both patients and dental team are regularly exposed to water and aerosols generated by dental equipment. Studies have demonstrated that DUWLs provide a favourable environment for microbial proliferation and biofilm formation, and that water is consequently often contaminated with high densities of various microorganisms (bacteria, fungi, protozoa, viruses). The presence of high levels of microbial contamination may be a health problem for dentists and patients, especially those who are immunocompromised. The current status of knowledge on microbial contamination of DUWLs is presented, with an emphasis on the infectious risk associated with DUWLs and on the various approaches for disinfecting and protecting DUWLs.
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Infecciones Bacterianas/transmisión , Instrumentos Dentales/microbiología , Micosis/transmisión , Infecciones por Protozoos/transmisión , Virosis/transmisión , Microbiología del Agua , Infecciones Bacterianas/microbiología , Biopelículas/crecimiento & desarrollo , Humanos , Micosis/microbiología , Infecciones por Protozoos/parasitología , Medición de Riesgo , Virosis/virologíaRESUMEN
The cloning of thrombopoietin together with advances in the culture of hematopoietic stem cells have paved the way for the study of megakaryopoiesis, ongoing clinical trials and, in the future, for the potential therapeutic use of ex vivo produced blood substitutes, such as platelets. This chapter describes a 14-day culture protocol for the production of human megakaryocytes (MKs) and platelets, and assays that can be used to characterize the functional properties of the platelets produced ex vivo. CD34(+) cells isolated from cord blood cells are grown in a serum-free medium supplemented with newly developed cytokine cocktails optimized for MK differentiation, expansion, and maturation. Detailed methodologies for flow cytometry analysis of MKs and platelets, for the purification of platelets and functional assays, are presented together with supporting figures. The chapter also provides a brief review on megakaryocytic differentiation and ex vivo MK cultures.
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Plaquetas/citología , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Megacariocitos/citología , Trombopoyesis , Separación Celular , Células Cultivadas , Citometría de Flujo , HumanosRESUMEN
The development of culture processes for hematopoietic progenitors could lead to the development of a complementary source of platelets for therapeutic purposes. However, functional characterization of culture-derived platelets remains limited, which raises some uncertainties about the quality of platelets produced in vitro. The aim of this study was to define the proportion of functional platelets produced in cord blood CD34+ cell cultures. Toward this, the morphological and functional properties of culture-derived platelet-like particles (PLPs) were critically compared to that of blood platelets. Flow cytometry combined with transmission electron microscopy analyses revealed that PLPs formed a more heterogeneous population of platelets at a different stage of maturation than blood platelets. The majority of PLPs harbored the fibrinogen receptor αIIbß3, but a significant proportion failed to maintain glycoprotein (GP)Ibα surface expression, a component of the vWF receptor essential for platelet functions. Importantly, GPIbα extracellular expression correlated closely with platelet function, as the GPIIb+ GPIbα+ PLP subfraction responded normally to agonist stimulation as evidenced by α-granule release, adhesion, spreading, and aggregation. In contrast, the GPIIb+ GPIbαâ» subfraction was unresponsive in most functional assays and appeared to be metabolically inactive. The present study confirms that functional platelets can be generated in cord blood CD34+ cell cultures, though these are highly susceptible to ectodomain shedding of receptors associated with loss of function. Optimization of culture conditions to prevent these deleterious effects and to homogenize PLPs is necessary to improve the quality and yields of culture-derived platelets before they can be recognized as a suitable complementary source for therapeutic purposes.