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

Single-cell genomics has transformed our understanding of complex cellular systems. However, excessive costs and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems, and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the interpretation of functional capacities of such precisely mapped cell states at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

36The bone marrow (BM) constitutes the primary site for life-long blood production and skeletal 37 regeneration. However, its cellular composition and the spatial organization into distinct 38 'niches' remains controversial. Here, we combine single-cell and spatially resolved 39 transcriptomics to systematically map the molecular and cellular composition of the endosteal, 40 sinusoidal, and arteriolar BM niches. This allowed us to transcriptionally profile all major BM 41 resident cell types, determine their localization, and clarify the cellular and spatial sources of 42 key growth factors and cytokines. Our data demonstrate that previously unrecognized Cxcl12-43 abundant reticular (CAR) cell subsets (i.e. Adipo-and Osteo-CAR cells) differentially localize 44 to sinusoidal or arteriolar surfaces, locally act as 'professional cytokine secreting cells', and 45 thereby establish distinct peri-vascular micro-niches. Importantly, we also demonstrate that the 46 3-dimensional organization of the BM can be accurately inferred from single-cell gene 47 expression data using the newly developed RNA-Magnet algorithm. Together, our study reveals 48 the cellular and spatial organization of BM niches, and offers a novel strategy to dissect the 49 complex organization of whole organs in a systematic manner. 50 51 One Sentence Summary: Integration of single-cell and spatial transcriptomics reveals the 52 molecular, cellular and spatial organization of bone marrow niches 53 54 65 endosteal niches 4-10 . To gain a global understanding of cell types and niches in the BM, we 66 have generated a single-cell RNA-sequencing (scRNAseq)-based molecular map of all major 67 BM populations. We then used spatially resolved transcriptomics in combination with novel 68 3 computational tools to allocate cell types to different BM niches, determine molecular 69 mediators of intercellular interactions, and identify the cellular and spatial sources of niche 70 factors. 71 72 RESULTS 73 Identification and characterization of BM resident cell types by scRNAseq 74 Frequencies of BM cell types differ by several orders of magnitude 11 , imposing a challenge to 75 scRNAseq approaches. To capture both highly abundant as well as extremely rare BM resident 76 cells, we performed droplet-based scRNAseq 12 of cells from total mouse BM, followed by 77 progressive depletion of highly abundant cell types or enrichment of rare populations from 78 undigested BM or enzymatically digested bones (Figure 1a). In total, our dataset comprises 79 7497 cells with a median detection of 1999 genes per single cell, which formed 32 clusters 80 corresponding to distinct cell types or stages of differentiation (Figure 1b, S1). Importantly, this 81 map is not quantitative with regard to the relative size of the different cell populations, since 82 dissociation rates largely differ between cell types 11 . As detailed below, cell type annotation 83 was performed based on marker gene expression (Table S1, Figure S2,3), gene ontology 84 analyses (Table S1), and by quantifyin...

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Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, but the importance of the diencephalon in TCA mapping is unknown. We report that disruption of diencephalic development by Pax6 deletion results in a thalamocortical projection containing mapping errors. We used conditional mutagenesis to test whether these errors are due to the disruption of pioneer projections from prethalamus to thalamus and found that, although this correlates with abnormal TCA fasciculation, it does not induce topographical errors. To test whether the thalamus contains navigational cues for TCAs, we used slice culture transplants and gene expression studies. We found the thalamic environment is instructive for TCA navigation and that the molecular cues netrin 1 and semaphorin 3a are likely to be involved. Our findings indicate that the correct topographic mapping of TCAs onto the cortex requires the order to be established from the earliest stages of their growth by molecular cues in the thalamus itself.

The step-wise acquisition of genetic abnormalities in cancer is thought to represent a major driver of disease initiation, relapse and therapy resistance. Acute myeloid leukemia (AML) represents a prime example of an aggressive cancer that develops in a multi-step manner from multipotent hematopoietic progenitors via pre-leukemic intermediates to leukemic cells. While bulk and single-cell genomics provide powerful tools to study the phylogenetics of cancer evolution, the specific transcriptomic changes induced by the accumulation of mutations remain largely unexplored. Here, we introduce MutaSeq, a combined single-cell genetic and transcriptomics platform for the identification of molecular consequences of cancer evolution. Through in-depth profiling of an AML patient, we demonstrate that MutaSeq is capable of: (1) fine-mapping clonal and developmental hierarchies (2) quantifying the ability of leukemic and pre-leukemic clones to give rise to mature lineages and (3) identifying surface markers and mRNA transcripts specific to pre-leukemic, leukemic, and residual healthy cells. The experimental and analytical approach presented here is broadly applicable to other types of cancer, and can help identify targets for eradicating both pre-cancerous and cancerous reservoirs of relapse.