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BACKGROUND: Language barriers were reported to affect timely access to health care and outcome. The aim of this study was to investigate the effect of language disparity on quality benchmarks of acute ischemic stroke therapy. METHODS: Consecutive patients with acute ischemic stroke at the University of California Irvine Medical Center from 2013 to 2016 were studied. Patients were categorized into 3 groups according to their preferred language: English, Spanish, and other languages. Quality benchmarks and outcomes of the 3 language groups were analyzed. RESULTS: Of the 928 admissions, 69.7% patients recorded English as preferred language, as compared to 17.3% Spanish and 13.0% other languages. There was no significant difference in the rate of receiving intravenous thrombolysis (24.3, 22.1 and 21.0%), last-known-well to door time, door-to-imaging time, door-to-needle time, and hospital length of stay among the 3 language groups. In univariate analysis, the other languages group had lower chance of favorable outcomes than the English-speaking group (26.3% vs 40.4, p < 0.05) while the Spanish-speaking group had lower mortality rate than English-speaking group (3.1% vs 7.7%, p = 0.05). After adjusting for age and initial NIHSS scores, multivariate regression models showed no significant difference in favorable outcomes and mortality between different language groups. CONCLUSION: We demonstrate no significant difference in quality benchmarks and outcome of acute ischemic stroke among 3 different language groups. Our results suggest that limited English proficiency is not a significant barrier for time-sensitive stroke care at Comprehensive Stroke Center.
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AVC Isquêmico/tratamento farmacológico , Idioma , Terapia Trombolítica , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Neural stem and progenitor cells (NSPCs) are an extremely important group of cells that form the central nervous system during development and have the potential to repair damage in conditions such as stroke impairment, spinal cord injury and Parkinson's disease degradation. Current schemes for separation of NSPCs are inadequate due to the complexity and diversity of cells in the population and lack sufficient markers to distinguish diverse cell types. This study presents an unbiased high-resolution separation and characterization of NSPC subpopulations using direct current insulator-based dielectrophoresis (DC-iDEP). The properties of the cells were identified by the ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. The ratio factor of NSPCs showed more heterogeneity variance (SD = 3.4-3.9) than the controlled more homogeneous human embryonic kidney cells (SD = 1.1), supporting the presence of distinct subpopulations of cells in NSPC cultures. This measure reflected NSPC fate potential since the ratio factor distribution of more neurogenic populations of NSPCs was distinct from the distribution of astrogenic NSPC populations (confidence level >99.9%). The abundance of NSPCs captured with different ranges of ratio of EK to DEP mobilities also exhibit final fate trends consistent with established final fates of the chosen samples. DC-iDEP is a novel, label-free and non-destructive method for differentiating and characterizing, and potentially separating, neural stem cell subpopulations that differ in fate.
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Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology.
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Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Complemento C1q/biossíntese , Complemento C3a/biossíntese , Células-Tronco Neurais/fisiologia , Neutrófilos/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Complemento C1q/antagonistas & inibidores , Complemento C1q/genética , Complemento C1q/imunologia , Complemento C3a/antagonistas & inibidores , Complemento C3a/genética , Complemento C3a/imunologia , Meios de Cultivo Condicionados , Humanos , Imunidade Inata , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/imunologia , Neutrófilos/imunologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Whole cell membrane capacitance is an electrophysiological property of the plasma membrane that serves as a biomarker for stem cell fate potential. Neural stem and progenitor cells (NSPCs) that differ in ability to form neurons or astrocytes are distinguished by membrane capacitance measured by dielectrophoresis (DEP). Differences in membrane capacitance are sufficient to enable the enrichment of neuron- or astrocyte-forming cells by DEP, showing the separation of stem cells on the basis of fate potential by membrane capacitance. NSPCs sorted by DEP need not be labeled and do not experience toxic effects from the sorting procedure. Other stem cell populations also display shifts in membrane capacitance as cells differentiate to a particular fate, clarifying the value of sorting a variety of stem cell types by capacitance. Here, we describe methods developed by our lab for separating NSPCs on the basis of capacitance using several types of DEP microfluidic devices, providing basic information on the sorting procedure as well as specific advantages and disadvantages of each device.
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Diferenciação Celular , Separação Celular/métodos , Células-Tronco Neurais/citologia , Neurônios/citologia , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Capacitância Elétrica , Eletroforese , Dispositivos Lab-On-A-ChipRESUMO
Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. We found that the mechanically gated ion channel Piezo1 is expressed by brain-derived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca(2+), a known modulator of differentiation, in a substrate-stiffness-dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells.
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Sinalização do Cálcio/fisiologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Técnicas de Silenciamento de Genes , Humanos , Canais Iônicos/genética , Masculino , Células-Tronco Multipotentes/citologia , Células-Tronco Neurais/citologiaRESUMO
AIM: To synthesize evidence from systematic reviews on the management of urinary incontinence and promotion of continence using conservative/behavioural approaches in older people in care homes to inform clinical practice, guidelines and research. BACKGROUND: Incontinence is highly prevalent in older people in care home populations. DESIGN: Systematic review of systematic reviews with narrative synthesis. DATA SOURCES: Electronic searches of published systematic reviews in English using MEDLINE and CINAHL with no date restrictions up to September 2013. Searches supplemented by hand searching and electronic searching of Cochrane Library and PROSPERO. REVIEW METHODS: PRISMA statement was followed, as were established methods for systematic review of systematic reviews. RESULTS: Five systematic reviews of high quality were included, three specific to intervention studies and two reviewed descriptive studies. Urinary incontinence was the primary outcome in three reviews with factors associated with the management of urinary incontinence the primary outcome for the other reviews. CONCLUSION: Toileting programmes, in particular prompted voiding, with use of incontinence pads are the main conservative behavioural approach for the management of incontinence and promotion of continence in this population with evidence of effectiveness in the short term. Evidence from associated factors; exercise, mobility, comorbidities, hydration, skin care, staff perspectives, policies and older people's experiences and preference are limited. The majority of evidence of effectiveness are from studies from one country which may or may not be transferable to other care home populations. Future international studies are warranted of complex combined interventions using mixed methods to provide evidence of effectiveness, context of implementation and economic evaluation.
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Promoção da Saúde , Casas de Saúde/organização & administração , Incontinência Urinária/enfermagem , Micção , Idoso , Feminino , Humanos , MasculinoRESUMO
AIM: Review of intervention studies of associated factors with incontinence as the primary outcome in older people in care homes to identify and inform practice and future research. BACKGROUND: Incontinence is highly prevalent among care home populations. Previous reviews of descriptive and intervention studies have used urinary incontinence as the primary outcome. DESIGN: Systematic review and narrative summary. DATA SOURCES: Electronic searches of English empirical studies undertaken using MEDLINE and CINAHL from January 1966-May 2010. All relevant empirical designs were selected from MEDLINE highly sensitive search strings from the Cochrane Incontinence Review Group, modified to exclude surgical and pharmacological studies REVIEW METHODS: The PRISMA statement was followed and established methods for systematic review to produce a narrative summary. RESULTS: Nine studies identified relating to associated factors with the management of incontinence in care homes. Factors included economic data, skin care, exercise studies, staff quality and prompted voiding adherence and the promotion of continence by the management of dehydration and incontinence. CONCLUSION: Managing incontinence and promoting continence in care homes is complex, requiring time and cost-efficient management procedures to contain the problem and deliver quality, achievable care. When developing and designing systems of care in care homes, it is important to also recognize the impact of associated factors. As with any healthcare intervention programme, resources are required to implement the protocols. Economic evaluation studies are limited, with further studies warranted alongside preventative studies to maintain long-term continence in these populations.
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Incontinência Fecal/enfermagem , Incontinência Urinária/enfermagem , Idoso , HumanosRESUMO
Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%-50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3' untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , Poliadenilação , RNA Mensageiro/química , Análise de Sequência de RNA/métodos , Animais , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Células HeLa , Histonas/química , Humanos , Camundongos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genéticaRESUMO
Undifferentiated neural stem and progenitor cells (NSPCs) encounter extracellular signals that bind plasma membrane proteins and influence differentiation. Membrane proteins are regulated by N-linked glycosylation, making it possible that glycosylation plays a critical role in cell differentiation. We assessed enzymes that control N-glycosylation in NSPCs and found that loss of the enzyme responsible for generating ß1,6-branched N-glycans, N-acetylglucosaminyltransferase V (MGAT5), led to specific changes in NSPC differentiation in vitro and in vivo. Mgat5 homozygous null NSPCs in culture formed more neurons and fewer astrocytes compared with wild-type controls. In the brain cerebral cortex, loss of MGAT5 caused accelerated neuronal differentiation. Rapid neuronal differentiation led to depletion of cells in the NSPC niche, resulting in a shift in cortical neuron layers in Mgat5 null mice. Glycosylation enzyme MGAT5 plays a critical and previously unrecognized role in cell differentiation and early brain development.
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Encéfalo , Proteínas de Membrana , Neurogênese , Animais , Camundongos , Encéfalo/crescimento & desenvolvimento , Glicosilação , Camundongos KnockoutRESUMO
PURPOSE: To survey recently graduated European ophthalmologists concerning cataract surgery (CS) training opportunities. SETTING: Countries affiliated to the European Board of Ophthalmology (EBO). DESIGN: Cross-sectional study of anonymous survey results. METHODS: A 23-question online survey was emailed to candidates who sat the EBO Diploma Examination as residents between 2018 and 2022. RESULTS: 821 ophthalmologists from 30 countries completed the survey. The mean residency duration was 4.73 (SD 0.9) years. The mean reported number of entire CS procedures performed was 80.7 (SD 100.6) at the end of residency, but more than 25% of respondents (n = 210) had received no live CS training during their residency. The self-confidence (scale, 1 to 10) to perform a simple case or challenging case, manage posterior capsular rupture, and realize a corneal stitch were rated 4.1, 3.2, 4.2, 2.4, respectively. We observed extensive variation in clinical exposure to CS and self-reported confidence to perform CS between European trainees. Females reported a mean of 18% fewer entire procedures than their male colleagues and were also less confident in their surgical skills (P < .05). Trainees in residency programs longer than 5 years performed fewer procedures and were less confident than trainees in residences of shorter duration (P < .001). The importance of fellowships to complete surgical education was rated 7.7 out of 10. CONCLUSIONS: CS training across European countries lacks harmony. Female ophthalmology trainees continue, as in other specialties, to experience apparent gender bias. European level recommendations seem necessary to raise and harmonize competency-based CS training programs and promote post-residency fellowship training programs.
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Extração de Catarata , Catarata , Internato e Residência , Oftalmologia , Feminino , Humanos , Masculino , Competência Clínica , Estudos Transversais , Educação de Pós-Graduação em Medicina/métodos , Europa (Continente) , Oftalmologia/educação , Sexismo , Inquéritos e Questionários , Extração de Catarata/educaçãoRESUMO
AIM: This is a review of descriptive studies with incontinence as the primary focus in older people in care homes. BACKGROUND: Incontinence is prevalent among residents of care home populations. DATA SOURCES: MEDLINE and CINAHL were searched from 1996 to 2007 using the highly sensitive search strings of the Cochrane Incontinence Review Group for urinary and faecal incontinence including all research designs. Search strings were modified to enhance selectiveness for care homes and older people and exclude studies involving surgical or pharmacological interventions. Searching of reference sections from identified studies was also used to supplement electronic searches. The Cochrane Library was searched for relevant systematic reviews to locate relevant studies from those included or excluded from reviews. The search was limited to English-language publications. METHODS: A systematic review of studies on the management of incontinence, promotion of continence or maintenance of continence in care homes was conducted in 2007-2009. This is a report of descriptive studies. Results. Ten studies were identified that reported on prevalence and incidence of incontinence (urinary with or without faecal), policies, assessment, documentation, management or economic evaluation of its management. Use of incontinence pads and toileting programmes comprised the most common management approaches used. No studies were identified that attempted to maintain continence of residents in care homes. CONCLUSIONS: Studies on maintaining continence and identifying components of toileting programmes that are successful in managing or preventing incontinence and promoting continence in residents of care home populations along with their economic evaluation are warranted.
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Incontinência Fecal/terapia , Promoção da Saúde , Instituição de Longa Permanência para Idosos/organização & administração , Pesquisa em Enfermagem , Incontinência Urinária/terapia , Atividades Cotidianas , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Incontinência Fecal/epidemiologia , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Tampões Absorventes para a Incontinência Urinária/estatística & dados numéricos , Masculino , Prontuários Médicos/normas , Avaliação em Enfermagem , Planejamento de Assistência ao Paciente/normas , Guias de Prática Clínica como Assunto , Prevalência , Treinamento no Uso de Banheiro , Incontinência Urinária/economia , Incontinência Urinária/epidemiologiaRESUMO
Polymeric scaffolds formed from synthetic or natural materials have many applications in tissue engineering and medicine, and multiple material properties need to be optimized for specific applications. Recent studies have emphasized the importance of the scaffolds' mechanical properties to support specific cellular responses in addition to considerations of biochemical interactions, material transport, immunogenicity, and other factors that determine biocompatibility. Fibrin gels formed from purified fibrinogen and thrombin, the final two reactants in the blood coagulation cascade, have long been shown to be effective in wound healing and supporting the growth of cells in vitro and in vivo. Fibrin, even without additional growth factors or other components has potential for use in neuronal wound healing in part because of its mechanical compliance that supports the growth of neurons without activation of glial proliferation. This review summarizes issues related to the use of fibrin gels in neuronal cell contexts, with an emphasis on issues of immunogenicity, and considers the potential advantages and disadvantages of fibrin prepared from non-mammalian sources.
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Materiais Biocompatíveis/metabolismo , Sistema Nervoso Central/lesões , Fibrina , Géis , Cicatrização/efeitos dos fármacos , Animais , Materiais Biocompatíveis/química , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Sistema Nervoso Central/fisiologia , Fibrina/química , Fibrina/metabolismo , Fibrinogênio/metabolismo , Géis/química , Géis/farmacologia , Humanos , Teste de Materiais , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Trombina/metabolismo , Alicerces TeciduaisRESUMO
Stem cells have attracted significant attention due to their regenerative capabilities and their potential for the treatment of disease. Consequently, significant research effort has focused on the development of protein- and polypeptide-based materials as stem cell substrates and scaffolds. Here, we explore the ability of reflectin, a cephalopod structural protein, to support the growth of murine neural stem/progenitor cells (mNSPCs). We observe that the binding, growth, and differentiation of mNSPCs on reflectin films is comparable to that on more established protein-based materials. Moreover, we find that heparin selectively inhibits the adhesion of mNSPCs on reflectin, affording spatial control of cell growth and leading to a >30-fold change in cell density on patterned substrates. The described findings highlight the potential utility of reflectin as a stem cell culture material.
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Cefalópodes , Células-Tronco Neurais , Animais , Diferenciação Celular , Proliferação de Células , Camundongos , ProteínasRESUMO
Human neural stem and progenitor cells (hNSPCs) have therapeutic potential to treat neural diseases and injuries since they provide neuroprotection and differentiate into astrocytes, neurons, and oligodendrocytes. However, cultures of hNSPCs are heterogeneous, containing cells linked to distinct differentiated cell fates. HNSPCs that differentiate into astrocytes are of interest for specific neurological diseases, creating a need for approaches that can detect and isolate these cells. Astrocyte-biased hNSPCs differ from other cell types in electrophysiological properties, namely membrane capacitance, and we hypothesized that this could be used to enrich these cells using dielectrophoresis (DEP). We implemented a two-step DEP sorting scheme, consisting of analysis to define the optimal sorting frequency followed by separation of cells at that frequency, to test whether astrocyte-biased cells could be separated from the other cell types present in hNSPC cultures. We developed a novel device that increased sorting reproducibility and provided both enriched and depleted cell populations in a single sort. Astrocyte-biased cells were successfully enriched from hNSPC cultures by DEP sorting, making this the first study to use electrophysiological properties for label-free enrichment of human astrocyte-biased cells. Enriched astrocyte-biased human cells enable future experiments to determine the specific properties of these important cells and test their therapeutic efficacy in animal models of neurological diseases.
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Separação Celular/instrumentação , Dispositivos Lab-On-A-Chip , Células-Tronco Neurais/citologia , Astrócitos/citologia , Técnicas Biossensoriais/instrumentação , Linhagem Celular , Capacitância Elétrica , Desenho de Equipamento , Humanos , Neurônios/citologia , Oligodendroglia/citologiaRESUMO
Modeling the in vivo microenvironment typically involves placing cells in a three-dimensional (3D) extracellular matrix (ECM) in physiologically relevant context with respect to other cells. The mechanical and chemical features of 3D microenvironments play important roles in tissue engineering, tumor growth and metastasis, and in defining stem cell niches, and it is increasingly recognized that cells behave much differently when surrounded by a 3D ECM than when anchored to a 2D substrate. To create microenvironments that more closely mimic in vivo settings, here we describe a novel microfluidic device that allows multiple discrete constructs of 3D cell-laden hydrogels to be patterned in a sequence of simple steps. The microfluidic platform allows for real-time imaging of the interactions between multiple cell types exposed to both autocrine and paracrine signaling molecules, all within a 3D ECM environment. Detailed modeling determined that surface tension, hydrophobic interactions, and spatial geometry were important factors in containing the gels within distinct separate channels during the filling process. This allowed us to pattern multiple gel types side-by-side and pattern 3D gels spatially with tight dimensional control. Cells embedded in gels could be patterned by culturing MDA-MB-231 metastatic breast cancer cells and RAW 264.1 macrophage cells within distinct collagen type I and Matrigel ECM environments, respectively. Over a 7 day culture experiment, RAW cells invaded into neighboring gels containing MDA-MB-231 cells, but not into gels lacking cells. These studies demonstrate the versatility and potential of this new microfluidic platform to engineer 3D microscale architectures to investigate cell-cell and cell-matrix interactions.
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Técnicas de Cocultura/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Comunicação Celular , Linhagem Celular Tumoral , Colágeno/metabolismo , Desenho de Equipamento , Matriz Extracelular/metabolismo , Humanos , Injeções , Pressão , Tensão Superficial , Integração de Sistemas , Fatores de Tempo , Engenharia TecidualRESUMO
This paper presents a novel design and separation strategy for lateral flow-through separation of cells/particles in microfluidics by dual frequency coupled dielectrophoresis (DEP) forces enabled by vertical interdigitated electrodes embedded in the channel sidewalls. Unlike field-flow-fractionation-DEP separations in microfluidics, which utilize planar electrodes on the microchannel floor to generate a DEP force to balance the gravitational force and separate objects at different height locations, lateral separation is enabled by sidewall interdigitated electrodes that are used to generate non-uniform electric fields and balanced DEP forces along the width of the microchannel. In the current design, two separate AC electric fields are applied to two sets of independent interdigitated electrode arrays fabricated in the sidewalls of the microchannel to generate differential DEP forces that act on the cells/particles flowing through. Individual particles (cells or beads) will experience DEP forces differently due to the difference in their dielectric properties. The balance of the differential DEP forces from the electrode arrays will position dissimilar particles at distinct equilibrium planes across the width of the channel. When coupled with fluid flow, this results in lateral separation along the width of the microchannel and the separated particles can thus be automatically directed into branched channel outlets leading to different reservoirs for downstream processing. In this paper, we present the design and analysis of lateral separation enabled by dual frequency coupled DEP, and cell/bead and cell/cell separations are demonstrated with this lateral separation strategy. With vertical interdigitated electrodes on the sidewall, the height of the microchannel can be increased without losing the electric field strength in contrast to other multiple frequency DEP devices with planar electrodes. As a result, populations of cells can be separated simultaneously instead of one by one to enable high-throughput sorting microfluidic devices.
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Separação Celular/instrumentação , Eletroforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Algoritmos , Linhagem Celular , Separação Celular/métodos , Simulação por Computador , Eletrodos , Eletroforese/métodos , Desenho de Equipamento , Humanos , Técnicas Analíticas Microfluídicas/métodos , MicroesferasRESUMO
The relatively new field of stem cell biology is hampered by a lack of sufficient means to accurately determine the phenotype of cells. Cell-type-specific markers, such as cell surface proteins used for flow cytometry or fluorescence-activated cell sorting, are limited and often recognize multiple members of a stem cell lineage. We sought to develop a complementary approach that would be less dependent on the identification of particular markers for the subpopulations of cells and would instead measure their overall character. We tested whether a microfluidic system using dielectrophoresis (DEP), which induces a frequency-dependent dipole in cells, would be useful for characterizing stem cells and their differentiated progeny. We found that populations of mouse neural stem/precursor cells (NSPCs), differentiated neurons, and differentiated astrocytes had different dielectric properties revealed by DEP. By isolating NSPCs from developmental ages at which they are more likely to generate neurons, or astrocytes, we were able to show that a shift in dielectric property reflecting their fate bias precedes detectable marker expression in these cells and identifies specific progenitor populations. In addition, experimental data and mathematical modeling suggest that DEP curve parameters can indicate cell heterogeneity in mixed cultures. These findings provide evidence for a whole cell property that reflects stem cell fate bias and establish DEP as a tool with unique capabilities for interrogating, characterizing, and sorting stem cells.
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Diferenciação Celular , Células-Tronco/citologia , Animais , Astrócitos/citologia , Linhagem Celular , Linhagem da Célula , Separação Celular , Sobrevivência Celular , Desenvolvimento Embrionário , Humanos , Camundongos , Microfluídica , Neurônios/citologia , Eletricidade EstáticaRESUMO
We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort.
RESUMO
Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of "outside-in" mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues.