Cell signaling pathways involved in hematopoietic stem cell specification.

Generation of HSCs for regenerative medicine Hematopoietic stem cells (HSCs) are self-renewing, tissue-specific stem cells that give rise to all mature blood cell types. The capacity of HSCs to reconstitute the entire adult hematopoietic system after transplantation makes them invaluable for the treatment of various blood disorders. A significant limitation of this treatment is the need for immune compatibility between donor and host, thus there has always been an acute need for reliable cultivation or generation of HSCs. The recently demonstrated ability to generate induced pluripotent stem (iPS) cells that resemble embryonic stem cells (ESCs) now make generation of HSCs from stem cells a realistic goal. To date, pluripotent stem cells have been instructed by a variety of experimental approaches to recapitulate waves of hematopoiesis such as primitive and transient definitive cells [1], myelomonocytic cells [2], and multilineage progenitors with lymphoid potential [3] (Figure 1A). Surprisingly, concerted efforts to generate functional HSCs in vitro from pluripotent stem cells have thus far proven unsuccessful, indicating that our understanding of de novo generation of HSCs is insufficient [4] (Figure 1B). Therefore, it is crucial to precisely characterize the mechanisms of cell signaling events that occur in vivo to form functional HSCs. Importantly, recent studies mapping the process of HSC generation in vertebrate embryos demonstrated that HSCs emerge from hemogenic endothelium present in the floor of the dorsal aorta (DA) [5–9]. For this reason, the generation of hemogenic endothelium likely represents a critical prerequisite for successfully generating HSCs in vitro. While many major cell-signaling pathways conserved throughout the animal kingdom have been demonstrated as requirements for DA and/or HSC formation, the molecular mechanisms that each required effector molecule exerts in this context is unclear. In this chapter we summarize the roles of select cell-signaling pathways in HSC generation in the embryo and provide perspective on the in vitro instruction of HSCs fate for use in regenerative medicine. Figure 1 Pathways to hematopoietic differentiation in vitro and in vivo Hematopoietic stem cell emergence in the vertebrate embryo The HSCs that maintain homeostasis of the adult hematopoietic system are generated during embryogenesis, but are not the first blood cells to be formed in the embryo. HSC emergence is preceded by primitive and definitive waves that are defined by limited differentiation potentials. Primitive myeloid and erythroid cells are the first hematopoietic cells to emerge, but unlike adult blood progenitors, do not possess multilineage potential or the capacity to self-renew [10, 11] (Figure 1C). Following these primitive waves, the first transient definitive progenitors arise that possess multipotent erythromyeloid potential (EMPs) [10, 12, 13]. EMPs are similar to HSCs in that they have multilineage potential, but are separated by the fact that they do not possess lymphoid potential or the capacity to appreciably self-renew (Figure 1D). The anatomical sites of emergence from which these waves arise vary according to species as shown by transplantation, imaging, and lineage tracing studies. The first three hematopoietic waves are found in the yolk sac in mammals and birds, in anterior/posterior ventral blood islands in frogs, and anterior/posterior lateral mesoderm in fish, the details of which are reviewed elsewhere [14, 15]. In contrast, HSCs emerge from hemogenic endothelium within the floor of the dorsal aorta in a process termed endothelial to hematopoietic transition (EHT) in all vertebrate species analyzed [6, 7, 9, 16] (Figure 1E). Nascent HSCs have been defined by their capacity in mammals to long-term reconstitute immune-deficient adult recipients and colonize adult hematopoietic organs [17, 18]. Developmental hematopoiesis thus progresses through four ordered waves, the last of which generates HSCs. A major question regarding the ontogeny of the hematopoetic system is how different regions of mesodermal derivatives are specified into each of these related but distinct fates. Transplantation experiments performed in frogs demonstrate that mesodermal precursors to blood cells experience bipotency for either primitive or definitive fate potential until the neurula stage [19], indicating that environmental signals are important during key stages of programming to different fate outcomes. Many of the signaling molecules involved in HSC specification are dispensable for other hematopoietic waves, indicating that the combined inputs that bestow HSC potential may be specific. Here we highlight the major signaling pathways involved in HSC emergence that are conserved across vertebrates.