The bone marrow (BM) constitutes the primary site for life-long blood production and skeletal regeneration. However, its cellular composition and the spatial organization into distinct 'niches' remains controversial. Here, we combine single-cell and spatially resolved transcriptomics to systematically map the molecular and cellular composition of the endosteal, sinusoidal, and arteriolar BM niches. This allowed us to transcriptionally profile all major BM resident cell types, determine their localization, and clarify the cellular and spatial sources of key growth factors and cytokines. Our data demonstrate that previously unrecognized Cxcl12abundant reticular (CAR) cell subsets (i.e. Adipo-and Osteo-CAR cells) differentially localize to sinusoidal or arteriolar surfaces, locally act as 'professional cytokine secreting cells', and thereby establish distinct peri-vascular micro-niches. Importantly, we also demonstrate that the 3-dimensional organization of the BM can be accurately inferred from single-cell gene expression data using the newly developed RNA-Magnet algorithm. Together, our study reveals the cellular and spatial organization of BM niches, and offers a novel strategy to dissect the complex organization of whole organs in a systematic manner.One Sentence Summary: Integration of single-cell and spatial transcriptomics reveals the molecular, cellular and spatial organization of bone marrow niches

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Sinusoidal endothelial cells and mesenchymal CXCL12-abundant reticular cells are principal bone marrow stromal components, which critically modulate haematopoiesis at various levels, including haematopoietic stem cell maintenance. These stromal subsets are thought to be scarce and function via highly specific interactions in anatomically confined niches. Yet, knowledge on their abundance, global distribution and spatial associations remains limited. Using three-dimensional quantitative microscopy we show that sinusoidal endothelial and mesenchymal reticular subsets are remarkably more abundant than estimated by conventional flow cytometry. Moreover, both cell types assemble in topologically complex networks, associate to extracellular matrix and pervade marrow tissues. Through spatial statistical methods we challenge previous models and demonstrate that even in the absence of major specific interaction forces, virtually all tissue-resident cells are invariably in physical contact with, or close proximity to, mesenchymal reticular and sinusoidal endothelial cells. We further show that basic structural features of these stromal components are preserved during ageing.

Spatially confined green-to-red photoconversion of fluorescent proteins with high-power, pulsed laser illumination is negligible, thus precluding optical selection of single cells in vivo. We report primed conversion, in which low-power, dual-wavelength, continuous-wave illumination results in pronounced photoconversion. With a straightforward addition to a conventional confocal microscope, we show confined primed conversion in living zebrafish and reveal the complex anatomy of individual neurons packed between neighboring cells.

Highlights d Transcriptional profiling of bone marrow stromal cells throughout postnatal lifespan d In situ validation of novel stromal markers to map the localization of PaS cells d Dynamic remodeling of stromal transcriptome in transition from juvenile to adult stages d Aging induces prototypical inflammatory transcriptional programs in stromal cells

Ageing is associated with impaired hematopoietic and immune function. This is caused in part by decreased hematopoietic stem cell (HSC) population fitness and an increased myeloid differentiation bias. The reasons for this aging-associated HSC impairment are incompletely understood. We here demonstrate that aged specific pathogen free (SPF) wild-type mice in contrast to young SPF mice produce more IL-1a/b in steady-state bone marrow (BM), with most of IL-1a/b being derived from myeloid BM cells. Further, blood of steady-state aged SPF wild-type mice contains higher levels of microbe associated molecular patterns (MAMPs), specifically TLR4 and TLR8 ligands. Also, BM myeloid cells from aged mice produce more IL-1b in vitro, and aged mice show higher and more durable IL-1a/b responses upon LPS stimulation in vivo. To test if HSC ageing is driven via IL-1a/b, we evaluated HSCs from IL-1 receptor 1 (IL-1R1) knock-out mice. Indeed, aged HSCs from IL-1R1 knock-out mice show significantly mitigated ageing-associated inflammatory signatures. Moreover, HSCs from aged IL-1R1KO and also from germ-free mice maintain unbiased lympho-myeloid hematopoietic differentiation upon transplantation, thus resembling this functionality of young HSCs. Importantly, in vivo antibiotic suppression of microbiota or pharmacologic blockade of IL-1 signaling in aged wild-type mice was similarly sufficient to reverse myeloid biased output of their HSC populations. Collectively, our data defines the microbiome-IL-1/IL-1R1 axis as a key, self-sustaining, but also therapeutically partially reversible driver of HSC inflamm-ageing.

Bone marrow (BM) stromal cells provide the structural and regulatory framework for hematopoiesis and contribute to developmental-stage specific niches, such as those preserving hematopoietic stem cell (HSCs). Despite recent advances in our understanding of stromal composition and function, little is known on the dynamic transcriptional remodeling that this compartment undergoes over time and during adaptation to stress. Similarly, how molecular changes in stroma are linked to age-related modulation of hematopoiesis is poorly understood. Using RNA-sequencing, we performed a longitudinal comparison of the transcriptional profile of four principal mesenchymal and endothelial stromal subsets, namely CXCL12-abundant reticular (CARc), PDGFR-α + Sca-1 + , sinusoidal (SECs) and arterial endothelial cells (AECs), isolated from early postnatal, adult and aged mice. Our data i) provide molecular fingerprints defining novel, cell-specific anatomical and functional features ii) reveal radical reprogramming of pro-hematopoietic, immune and matrisomic transcriptional programs during the narrow temporal transition from juvenile to adult stages iii) demonstrate that homeostatic aging is characterized by a progressive and pronounced upregulation of pro-inflammatory gene-expression and loss of stromal cell fitness. By profiling in vivo responses of stromal cells to infection-mimicking agents, we finally demonstrate that transcriptomic pathways elicited by sterile inflammation are largely recapitulated during aging, thereby supporting the inflammatory basis of aging-related adaptations of BM hematopoietic function. Transcriptomic profiling of BM stroma Helbling et al.

Hematopoietic stem cells (HSCs) have been long proposed to reside in defined anatomical locations within bone marrow (BM) tissues in direct contact or close proximity to nurturing cell types. Imaging techniques that allow the simultaneous mapping of HSCs and interacting cell types have been central to the discovery of basic principles of these so-called HSC niches. Despite major progress in the field, a quantitative and comprehensive model of the cellular and molecular components that define these specialized microenvironments is lacking to date, and uncertainties remain on the preferential localization of HSCs in the context of complex BM tissue landscapes. Recent technological breakthroughs currently allow for the quantitative spatial analysis of BM cellular components with extraordinary precision. Here, we critically discuss essential technical aspects related to imaging approaches, image processing tools, and spatial statistics, which constitute the three basic elements of rigorous quantitative spatial analyses of HSC niches in the BM microenvironment.