Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow (BM) remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging techniques and computational modelling to analyse significant tridimensional associations among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal BM. These arterioles are ensheathed exclusively by rare NG2+ pericytes, distinct from sinusoid-associated LepR+ cells. Pharmacological or genetic activation of HSC cell cycle alters the distribution of HSCs from NG2+ peri-arteriolar niches to LepR+ peri-sinusoidal niches. Conditional depletion of NG2+ cells induces HSC cycling and reduces functional long-term repopulating HSCs in BM. These results thus indicate that arteriolar niches are indispensable to maintain HSC quiescence.

Summary The molecular complexity of the bone marrow (BM) microenvironment and its response to stress are incompletely understood, despite its key role in the regulation of hematopoiesis. Here we map the transcriptional landscape of BM vascular, perivascular, and osteoblast niche populations at single-cell resolution at both homeostasis and under stress hematopoiesis. This analysis revealed a previously unappreciated level of cellular heterogeneity within the BM niche, identified novel cellular subsets, and resolved cellular sources of pro-hematopoietic growth factors, chemokines, and membrane-bound ligands. Under conditions of stress, our studies revealed a significant transcriptional remodeling of these niche elements, including an adipocytic skewing of the perivascular cells. Among the stress-induced changes, we observed that vascular Notch ligand delta-like ligands (Dll1,4) were downregulated. In the absence of vascular Dll4, hematopoietic stem cells (HSC) prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the BM niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress, and illustrate the utility of single cell transcriptomic data in systematically evaluating the regulation of hematopoiesis by discrete niche populations.

The haematopoietic stem cell (HSC) microenvironment in the bone marrow, termed the niche, ensures haematopoietic homeostasis by controlling the proliferation, self-renewal, differentiation and migration of HSCs and progenitor cells at steady state and in response to emergencies and injury. Improved methods for HSC isolation, driven by advances in single-cell and molecular technologies, have led to a better understanding of their behaviour, heterogeneity and lineage fate, and of the niche cells and signals that regulate their function. Niche regulatory signals can be in the form of cell-bound or secreted factors and other local physical cues. A combination of technological advances in bone marrow imaging and genetic manipulation of crucial regulatory factors has enabled the identification of several candidate cell types regulating the niche, including both non-haematopoietic (e.g. perivascular mesenchymal stem and endothelial cells) and HSC-derived (e.g. megakaryocytes, macrophages and regulatory T cells), with better topographical understanding of HSC localization in the bone marrow. Here, we review advances in our understanding of HSC regulation by niches during homeostasis, ageing and malignancy, and discuss their implications for the development of therapies to rejuvenate aged HSCs or niches or to disrupt self-reinforcing malignant niches.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by overexpressing combinations of factors such as Oct4, Sox2, Klf4, and c-Myc. Reprogramming is slow and stochastic, suggesting the existence of barriers limiting its efficiency. Here we identify senescence as one such barrier. Expression of the four reprogramming factors triggers senescence by up-regulating p53, p16INK4a , and p21 CIP1. Induction of DNA damage response and chromatin remodeling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells.Supplemental material is available at http://www.genesdev.org.

In the bone marrow (BM), hematopoietic stem cells (HSCs) lodge in specialized microenvironments that tightly control their proliferative state to adapt to the varying needs for replenishment of blood cells while also preventing exhaustion1. All putative niche cells suggested thus far have a non-hematopoietic origin2-8. Thus, it remains unclear how feedback from mature cells is conveyed to HSCs to adjust proliferation. Here we show that megakaryocytes (Mk) can directly regulate HSC pool size. Three-dimensional whole-mount imaging revealed that endogenous HSCs are frequently located adjacent to Mk in a non-random fashion. Selective in vivo depletion of Mk resulted in specific loss of HSC quiescence and led to a marked expansion of functional HSCs. Gene expression analyses revealed that Mk were the source of chemokine C-X-C motif ligand 4 (Cxcl4, also named platelet factor 4, Pf4) in the BM and Cxcl4 injection reduced HSC numbers via increased quiescence. By contrast, Cxcl4−/− mice exhibited increased HSC numbers and proliferation. Combined use of whole-mount imaging and computational modelling was highly suggestive of a megakaryocytic niche capable of influencing independently HSC maintenance by regulating quiescence. Thus, these results indicate that a terminally differentiated HSC progeny contributes to niche activity by directly regulating HSC behavior.