Spatial mapping of cellular senescence: emerging challenges and opportunities.

Cellular senescence is a well-established driver of aging and age-related diseases. There are many challenges to mapping senescent cells in tissues such as the absence of specific markers and their relatively low abundance and vast heterogeneity. Single-cell technologies have allowed unprecedented characterization of senescence; however, many methodologies fail to provide spatial insights. The spatial component is essential, as senescent cells communicate with neighboring cells, impacting their function and the composition of extracellular space. The Cellular Senescence Network (SenNet), a National Institutes of Health (NIH) Common Fund initiative, aims to map senescent cells across the lifespan of humans and mice. Here, we provide a comprehensive review of the existing and emerging methodologies for spatial imaging and their application toward mapping senescent cells. Moreover, we discuss the limitations and challenges inherent to each technology. We argue that the development of spatially resolved methods is essential toward the goal of attaining an atlas of senescent cells.

A cellular taxonomy of the adult human spinal cord.

The mammalian spinal cord functions as a community of cell types for sensory processing, autonomic control, and movement. While animal models have advanced our understanding of spinal cellular diversity, characterizing human biology directly is important to uncover specialized features of basic function and human pathology. Here, we present a cellular taxonomy of the adult human spinal cord using single-nucleus RNA sequencing with spatial transcriptomics and antibody validation. We identified 29 glial clusters and 35 neuronal clusters, organized principally by anatomical location. To demonstrate the relevance of this resource to human disease, we analyzed spinal motoneurons, which degenerate in amyotrophic lateral sclerosis (ALS) and other diseases. We found that compared with other spinal neurons, human motoneurons are defined by genes related to cell size, cytoskeletal structure, and ALS, suggesting a specialized molecular repertoire underlying their selective vulnerability. We include a web resource to facilitate further investigations into human spinal cord biology.

An open source toolkit for repurposing Illumina sequencing systems as versatile fluidics and imaging platforms.

Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument, comprising an epifluorescence microscope with integrated fluidics, into an open platform for programmable applications without need for specialized engineering or software development expertise. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. Given the widespread availability of HiSeq 2500 platforms and the simplicity of the modifications required to repurpose these systems, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.

Spatially resolved transcriptomics and its applications in cancer.

Spatially resolved transcriptomics (SRT) offers the promise of understanding cells and their modes of dysfunction in the context of intact tissues. Technologies for SRT have advanced rapidly with a large number being published in recent years. Diverse methods for SRT produce data at widely varying depth, throughput, accessibility and cost. Many published SRT methods have been demonstrated only in their labs of origin, while others have matured to the point of commercialization and widespread availability. Here we review technologies for SRT, and their application in studies of tumor heterogeneity.

A core viral protein binds host nucleosomes to sequester immune danger signals.

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.