Anne E. Wiblin

Nuclear organisation is thought to be important in regulating gene expression. Here we investigate whether human embryonic stem cells (hES) have a particular nuclear organisation, which could be important for maintaining their pluripotent state. We found that whereas the nuclei of hES cells have a general gene-density-related radial organisation of chromosomes, as is seen in differentiated cells, there are also distinctive localisations for chromosome regions and gene loci with a role in pluripotency. Chromosome 12p, a region of the human genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p we find no overall change in nuclear chromosome position, but instead we detect a relocalisation of the OCT4 locus, to a position outside its chromosome territory. There is also a smaller proportion of centromeres located close to the nuclear periphery in hES cells compared to differentiated cells. We conclude that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency. Understanding this level of hES cell biology provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.

Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.

Tissues and organs are composed of distinct cell types that must operate in\nconcert to perform physiological functions. Efforts to create high-dimensional\nbiomarker catalogs of these cells are largely based on transcriptomic\nsingle-cell approaches that lack the spatial context required to understand\ncritical cellular communication and correlated structural organization. To\nprobe in situ biology with sufficient coverage depth, several multiplexed\nprotein imaging methods have recently been developed. Though these\nantibody-based technologies differ in strategy and mode of immunolabeling and\ndetection tags, they commonly utilize antibodies directed against protein\nbiomarkers to provide detailed spatial and functional maps of complex tissues.\nAs these promising antibody-based multiplexing approaches become more widely\nadopted, new frameworks and considerations are critical for training future\nusers, generating molecular tools, validating antibody panels, and harmonizing\ndatasets. In this perspective, we provide essential resources and key\nconsiderations for obtaining robust and reproducible multiplexed antibody-based\nimaging data compiling specialized knowledge from domain experts and technology\ndevelopers.\n