RESUMO
Regenerative medicine approaches utilizing stem cells offer a promising strategy to address tendinopathy, a class of common tendon disorders associated with pain and impaired function. Tendon progenitor cells (TPCs) are important in healing and maintaining tendon tissues. Here we provide a comprehensive single cell transcriptomic profiling of TPCs from three normal and three clinically classified tendinopathy samples in response to mechanical stimuli. Analysis reveals seven distinct TPC subpopulations including subsets that are responsive to the mechanical stress, highly clonogenic, and specialized in cytokine or growth factor expression. The single cell transcriptomic profiling of TPCs and their subsets serves as a foundation for further investigation into the pathology and molecular hallmarks of tendinopathy in mechanical stimulation conditions.
Assuntos
Perfilação da Expressão Gênica , Análise de Célula Única , Células-Tronco/metabolismo , Células-Tronco/patologia , Tendinopatia/genética , Tendinopatia/patologia , Tendões/patologia , Adolescente , Adulto , Proteínas de Transporte de Cátions/metabolismo , Ciclo Celular/genética , Células Clonais , Regulação da Expressão Gênica , Humanos , Inflamação/patologia , Masculino , Mitocôndrias/genética , Estresse Mecânico , Adulto JovemRESUMO
In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5-25 µm in diameter) within highly monodisperse double emulsions (35 µm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.
Assuntos
Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Técnicas Analíticas Microfluídicas , Análise de Célula Única , Animais , Encapsulamento de Células , Linhagem Celular , Camundongos , Tamanho da Partícula , Fenótipo , Propriedades de SuperfícieRESUMO
Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.
Assuntos
Sistemas CRISPR-Cas/genética , Reprogramação Celular/genética , Edição de Genes , Mutagênese Sítio-Dirigida/métodos , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição/genética , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Recent studies have suggested that metabolic surgery reduces cancer risk. This study aims to determine if incident cancer is associated with the extent of weight loss after Roux-en-Y gastric bypass (RYGB). METHODS: Patients at a large tertiary bariatric surgery center were retrospectively reviewed to identify patients with no history of cancer at the time of RYGB. Diagnoses in the electronic health record, a tumor registry, and chart review were used to identify postoperative incident solid organ cancer. The overall incidence of organ cancer was estimated using Kaplan-Meier analysis. The percent total body weight loss (%TWL) in the 48 months after surgery but prior to cancer was compared between those that developed organ cancer versus those that did not using repeated measures linear regression. RESULTS: The 2943 patients had a mean age of 45.6 years (SD = 11.1), 81 % were female, and a mean baseline body mass index (BMI) of 47.2 kg/m2 (SD = 7.9). Median follow-up after surgery was 3.8 years (range = [<1, 12]). Incident organ cancer developed and was verified in 54 of the 2943 patients (1.8 %). Kaplan-Meier estimates for cancer at 3, 5, and 10 years postsurgery were 1.3, 2.5, and 4.2 %. After adjusting for age, BMI, sex, diabetes, hypertension, and dyslipidemia, patients that developed organ cancer achieved less weight loss (-1.5 % TWL, 95 % CI = [-2.9 %, -0.1 %], p = 0.034). CONCLUSIONS: Greater weight loss after metabolic surgery may be associated with lower organ cancer risk.
Assuntos
Anastomose em-Y de Roux/estatística & dados numéricos , Derivação Gástrica/estatística & dados numéricos , Neoplasias/epidemiologia , Obesidade Mórbida/epidemiologia , Obesidade Mórbida/cirurgia , Redução de Peso/fisiologia , Adolescente , Adulto , Idoso , Anastomose em-Y de Roux/métodos , Índice de Massa Corporal , Feminino , Derivação Gástrica/métodos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
In this paper we present an automated system for simultaneous measurement of CO(2) concentration, delta(13)C and delta(18)O from small (<1 mL) air samples in a short period of time (approximately 1 hour). This system combines continuous-flow isotope ratio mass spectrometry (CF-IRMS) and gas chromatography (GC) with an inlet system similar to conventional dual-inlet methods permitting several measurement cycles of standard and sample air. Analogous to the dual-inlet method, the precision of this system increases with the number of replicate cycles measured. The standard error of the mean for a measurement with this system is 0.7 ppm for the CO(2) concentration and 0.05 per thousand for the delta(13)C and delta(18)O with four replicate cycles and 0.4 ppm and 0.03 per thousand respectively with nine replicate cycles. The mean offset of our measurements from NOAA/CMDL analyzed air samples was 0.08 ppm for the CO(2) concentration, 0.01 per thousand for delta(13)C and 0.00 per thousand for delta(18)O. A specific list of the parts and operation of the system is detailed as well as some of the applications for micrometeorological and ecophysiological applications.