Endothelial cells with arterial and venous properties segregate into CXCR4+ and CXCR4? fractions within CD73+ non-HE population [74]. production and expansion. Introduction DL-AP3 Advances in cellular reprogramming technologies have created alternative platforms for the production of blood cells through inducing pluripotency in somatic cells or by way of direct conversion of non-hematopoietic cells into blood cells [1C6]. Generating autologous hematopoietic stem cells (HSCs) from expandable cells that can be clonally selected, represents a promising approach for novel patient-specific gene therapies. Induced pluripotent stem cells (iPSCs) can be precisely genetically modified with designer endonucleases, including the CRISPR/Cas9 system, and subsequently single cell sorted to ensure homogeneity of genomic editing and to eliminate potential clones with deleterious off-target effects [7,8]. Alternatively, the generation of gene-targeted iPSC lines with only a single clonal event can be achieved using simultaneous reprogramming and CRISPR/Cas9 gene editing in somatic cells [9]. In addition, reprogramming non-hematopoietic somatic and mature blood cells to pluripotency and their DL-AP3 subsequent differentiation into hematopoietic stem cells/progenitors offers DL-AP3 a promising tool for modeling blood diseases [10], studying primitive leukemia cells [11], and drug discovery [12]. However, generation of blood cells with robust multilineage engraftment potential from human PSCs (iPSCs and embryonic stem cells; ESCs) remains a significant challenge [1C3]. Overcoming the inherent limitations in this process requires a complete understanding and recapitulation in a culture dish of the process of HSC formation in the embryo. Since blood formation from endothelium represents the key event during HSC emergence production of HSCs from human PSCs and emerging new concepts in blood and HSC generation by direct conversion from somatic cells. Specification of adult HSCs during embryonic development Elegant experiments by Dieterlen-Lie`vre and coworkers using homotypic grafting of quail embryos on chicken yolk sacs revealed embryo proper as a major source of hematopoietic stem cells (HSCs) [13,14], and eventually led to the identification of the para-aortic splanchnopeure/aorta-gonad-mesonephros (P-Sp/AGM) region as a major site of definitive hematopoiesis and HSC origin in vertebrates, including humans [15C19]. In aorta, HSCs mature in intra-aortic hematopoietic clusters (IAHC) budding from the endothelial lining of the dorsal aorta. IAHC was originally described more than a century ago by Dantschakoff [20]. Further studies by early embryologists revealed that IAHCs are restricted to the ventral portion of caudal aortic wall and could only be found at certain stages of embryonic development, but not in adults [21C25]. Based on careful microscopic analysis of serial tissue sections of IAHC, these studies provided compelling support for the idea that hematopoietic cells in aorta arise through transition of flat aortic endothelial cells into round hematopoietic cells. They also introduced the terminology hemogenic endothelium (HE) to define the specialized subset of endothelium with blood forming potential. Direct confirmation of blood formation from HE came relatively recently from lineage tracing in chicken embryo using Di-LDL labeling [26], and in mouse embryo using a VE-cadherin-driven Cre-recombinase [27]. In addition, real-time observations documented the gradual acquisition of hematopoietic morphology and phenotype by aortic endothelial cells [28C30]. Although the concept Rabbit Polyclonal to ERCC5 of HE was initially developed based on AGM studies, it became clear that endothelium DL-AP3 in other embryonic and extraembryonic sites also possess hemogenic potential. Among them are the vitelline and umbilical arteries [31,32], placenta [33], head vasculature [34], endocardium [35], and yolk sac vessels [36C39]. Transient waves of hematopoiesis preceding HSC stage While HSC function is required for establishing life-long hematopoiesis after birth, the metabolic and growth-promoting processes in the embryo are supported by transient hematopoietic progenitors that are formed prior to the emergence of HSCs (embryonic day (E) 10.5C11 in mouse). The first wave of hematopoiesis takes place in yolk sac (E7.5, in mouse) with the generation of large nucleated primitive red blood cells, macrophages and megakaryocytes [40C42]. These primitive hematopoietic progenitors are derived directly from the mesodermal precursor, hemangioblast, through endothelial intermediates [43C45]. Shortly thereafter (E8.25 in mouse), transient definitive erythro-myeloid progenitors (EMPs) are generated in yolk sac [40,46]. Cells with T and B lymphoid potentials also arise prior to HSC emergence in different hematopoietic sites including yolk sac, para-aortic splanchnopelura, viteline and umbilical arteries, and placenta (reviewed in [47,48]). Interestingly, similar to the AGM region, EMPs and lymphoid cells in yolk sac originate through EHT from endothelial cells lining nascent capillaries, arterial and venous vessels [36C39,49], thereby indicating that blood formation through endothelial intermediates is a central process during development of the entire hematopoietic system. Hematopoietic differentiation and engraftment of human pluripotent stem cells Over the past decade multiple studies have.