______________________________________________________________________ 2 Understanding human bone marrow (BM) pathophysiology in the context of myelotoxic stress induced by drugs, radiation, or genetic mutations is of critical importance in clinical medicine. However, study of these dynamic cellular responses is hampered by the inaccessibility of living BM in vivo. Here, we describe a vascularized human Bone Marrow-on-a-Chip (BM Chip) microfluidic culture device for modeling bone marrow function and disease states. The BM Chip is comprised of a fluidic channel filled with a fibrin gel in which patient-derived CD34+ cells and bone marrow-derived stromal cells (BMSCs) are co-cultured, which is separated by a porous membrane from a parallel fluidic channel lined by human vascular endothelium. When perfused with culture medium through the vascular channel, the BM Chip maintains human CD34+ cells and supports differentiation and maturation of multiple blood cell lineages over 1 month in culture. Moreover, it recapitulates human myeloerythroid injury responses to drugs and gamma radiation exposure, as well as key hematopoietic abnormalities found in patients with the genetic disorder, Shwachman-Diamond Syndrome (SDS). These data establish the BM Chip as a new human in vitro model with broad potential utility for studies of BM dysfunction.The human BM is the site where all adult blood cells originate and thus BM dysfunction causes significant patient morbidity and mortality. It is a major target of drug-and radiationrelated toxicities due to its high cell proliferation rates, and it is also affected by a variety of genetic disorders from congenital marrow failure syndromes to myeloid malignancies. While these abnormalities can be diagnosed and managed by monitoring peripheral blood counts, it is the proliferation and differentiation of hematopoietic cells in the marrow that is directly targeted in these disease states. Aside from invasive biopsies, there are no methods to study these responses in situ over time in human patients.Various in vitro culture methods for human hematopoietic cells have been described, including culturing CD34+ hematopoietic progenitors in suspension (including methylcellulose-3 based assays) 1,2 , on stromal cell monolayers (e.g. Dexter culture and assays to assess longterm culture-initiating cells and cobblestone area-forming cells) 3,4 , or in perfused devices that may include bone or artificial scaffolds seeded with various stromal cells 5-9 . These in vitro methods, along with an extensive body of work in animal models, have been used to gain fundamental insight into the biology of hematopoiesis and hematopoietic stem cells 1,2,10 .However, the translational application of human in vitro culture methods to modeling hematopoietic injury and disease has been limited. Methylcellulose-based colony forming assays remain the workhorse, especially in the pharmaceutical industry, despite inherent limitations in the ability to manipulate the culture (e.g. add or remove drug) after the initial setup...

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening we identified CD44 as a new marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta gonad mesonephros (AGM) region. This allowed us to provide a very detailed phenotypical and transcriptional profile for haemogenic endothelial cells, characterising them with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists (Smad6, Smad7 and Bmper) and a downregulation of genes related to glycolysis and the TCA cycle. Moreover, we demonstrated that by inhibiting the interaction between CD44 and its ligand hyaluronan we could block EHT, identifying a new regulator of HSPC development.