Liquid biopsy, particularly cell-free RNA (cfRNA), has emerged as a promising non-invasive diagnostic tool for various diseases, including cancer, due to its accessibility and the wealth of information it provides. A key area of interest is the composition and cellular origin of cfRNA in the blood and the alterations in the cfRNA transcriptomic landscape during carcinogenesis. Investigating these changes can offer insights into the manifestations of tissue alterations in the blood, potentially leading to more effective diagnostic strategies. However, the consistency of these findings across different studies and their clinical utility remains to be fully elucidated, highlighting the need for further research in this area.

Homeostatic temperature regulation is fundamental to mammalian physiology and is controlled by acute and chronic responses of local, endocrine and nervous regulators. Here, we report that loss of the heparan sulfate proteoglycan, syndecan-1, causes a profoundly depleted intradermal fat layer, which provides crucial thermogenic insulation for mammals. Mice without syndecan-1 enter torpor upon fasting and show multiple indicators of cold stress, including activation of the stress checkpoint p38α in brown adipose tissue, liver and lung. The metabolic phenotype in mutant mice, including reduced liver glycogen, is rescued by housing at thermoneutrality, suggesting that reduced insulation in cool temperatures underlies the observed phenotypes. We find that syndecan-1, which functions as a facultative lipoprotein uptake receptor, is required for adipocyte differentiation in vitro. Intradermal fat shows highly dynamic differentiation, continuously expanding and involuting in response to hair cycle and ambient temperature. This physiology probably confers a unique role for Sdc1 in this adipocyte sub-type. The PPARγ agonist rosiglitazone rescues Sdc1-/- intradermal adipose tissue, placing PPARγ downstream of Sdc1 in triggering adipocyte differentiation. Our study indicates that disruption of intradermal adipose tissue development results in cold stress and complex metabolic pathology.

Acinar cells make up the majority of all cells in the pancreas, yet the source of new acinar cells during homeostasis remains unknown. Using multicolor lineage-tracing and organoid-formation assays, we identified the presence of a progenitor-like acinar cell subpopulation. These cells have long-term self-renewal capacity, albeit in a unipotent fashion. We further demonstrate that binuclear acinar cells are terminally differentiated acinar cells. Transcriptome analysis of single acinar cells revealed the existence of a minor population of cells expressing progenitor markers. Interestingly, a gain of the identified markers accompanied by a transient gain of proliferation was observed following chemically induced pancreatitis. Altogether, our study identifies a functionally and molecularly distinct acinar subpopulation and thus transforms our understanding of the acinar cell compartment as a pool of equipotent secretory cells.

Single-cell mRNA sequencing (scRNA-seq) is a powerful method to identify and classify cell types and reconstruct differentiation trajectories within complex tissues, such as the developing human cortex. scRNA-seq data also enables the discovery of cell type-specific marker genes and genes that regulate developmental transitions. Here we provide a brief overview of how scRNA-seq has been shaping the study of human cortex development, and present ShinyCortex, a resource that brings together data from recent scRNA-seq studies of the developing cortex for further analysis. ShinyCortex is based in R and displays recently published scRNA-seq data from the human and mouse cortex in a comprehensible, dynamic and accessible way, suitable for data exploration by biologists.

Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.

Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.