Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity in health and disease. However, scRNA-seq requires lysing cells, which makes it impossible to link the individual cells to downstream molecular and phenotypic states. Here, we established Live-seq, an approach for single-cell transcriptome profiling that preserves cell viability during RNA extraction using fluidic force microscopy. Based on cell division, functional responses and whole-cell transcriptome read-outs, we show that Live-seq does not induce major cellular perturbations and therefore can function as a transcriptomic recorder. We demonstrate this recording capacity by preregistering the transcriptomes of individual macrophage-like RAW 264.7 cells that were subsequently subjected to time-lapse imaging after lipopolysaccharide (LPS) exposure. This enabled the unsupervised, genome-wide ranking of genes based on their ability to impact macrophage LPS response heterogeneity, revealing basal NFKBIA expression level and cell cycle state as major phenotypic determinants. Furthermore, we show that Live-seq can be used to sequentially profile the transcriptomes of individual macrophages before and after stimulation with LPS, thus enabling the direct mapping of a cell's trajectory. Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.

Adipose stem and precursor cells (ASPCs) give rise to adipocytes and determine the composition and plasticity of adipose tissue. Recently, several studies have demonstrated that ASPCs partition into at least three distinct cell subpopulations: Dpp4+ stem-like cells, Aoc3+ pre-adipocyte-like cells, and the enigmatic CD142+ cells. A great challenge now is to functionally characterize these distinct ASPC populations. Here, we focus on CD142+ ASPCs since discrepant properties have been assigned to this subpopulation, from adipogenic to non- and even anti-adipogenic. To address these inconsistencies, we comprehensively characterized mammalian subcutaneous CD142+ ASPCs across various sampling conditions. Our findings demonstrate that CD142+ ASPCs exhibit high molecular and phenotypic robustness, firmly supporting their non- and anti-adipogenic properties. However, these properties emerge in an age-dependent manner, revealing surprising temporal CD142+ ASPC behavioural alterations. Finally, using multi-omic and functional assays, we show that the inhibitory nature of these adipogenesis-regulatory CD142+ ASPCs (Aregs) is driven by specifically expressed secretory factors that cooperate with the retinoic acid signalling pathway to transform the adipogenic state of CD142- ASPCs into a non-adipogenic, Areg-like one.