Blood formation is believed to occur through step-wise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a “Continuum of LOw primed UnDifferentiated hematopoietic stem- and progenitor cells” (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady state haematopoiesis downstream of HSCs and provide a basis for the understanding of hematopoietic malignancies.

Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.

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