RESUMEN
Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity1. However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy2,3, thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.
Asunto(s)
Supervivencia Celular , Perfilación de la Expresión Génica , Macrófagos , RNA-Seq , Análisis de la Célula Individual , Transcriptoma , Tejido Adiposo/citología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Diferenciación Celular , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Genoma/efectos de los fármacos , Genoma/genética , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Inhibidor NF-kappaB alfa/genética , Especificidad de Órganos , Fenotipo , ARN/genética , ARN/aislamiento & purificación , RNA-Seq/métodos , RNA-Seq/normas , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodos , Análisis de Secuencia de ARN/normas , Análisis de la Célula Individual/métodos , Células del Estroma/citología , Células del Estroma/metabolismo , Factores de Tiempo , Transcriptoma/genéticaRESUMEN
Adipose tissue plasticity is orchestrated by molecularly and functionally diverse cells within the stromal vascular fraction (SVF). Although several mouse and human adipose SVF cellular subpopulations have by now been identified, we still lack an understanding of the cellular and functional variability of adipose stem and progenitor cell (ASPC) populations across human fat depots. To address this, we performed single-cell and bulk RNA sequencing (RNA-seq) analyses of >30 SVF/Lin- samples across four human adipose depots, revealing two ubiquitous human ASPC (hASPC) subpopulations with distinct proliferative and adipogenic properties but also depot- and BMI-dependent proportions. Furthermore, we identified an omental-specific, high IGFBP2-expressing stromal population that transitions between mesothelial and mesenchymal cell states and inhibits hASPC adipogenesis through IGFBP2 secretion. Our analyses highlight the molecular and cellular uniqueness of different adipose niches, while our discovery of an anti-adipogenic IGFBP2+ omental-specific population provides a new rationale for the biomedically relevant, limited adipogenic capacity of omental hASPCs.