It is thought that a

It is thought that a better understanding of the molecular and cellular mechanisms that regulate the emergence and maintenance of long-term repopulating hematopoietic stem liothyronine sodium (HSCs) during embryonic development would aid in the development of optimal protocols to generate such cells in vitro from PSCs. HSCs have been shown to emerge first from the aorta-gonad-mesonephros (AGM) region around embryonic day 10.5 (E10.5) in murine embryos (Medvinsky and Dzierzak, 1996). This occurs several days after the actual onset of hematopoietic activity, which is observed first in the yolk sac from E7.5 and next in the embryo proper from E9.0 (Palis et al., 1999). These early waves of hematopoiesis successively give rise to primitive erythroid, myeloid, definitive erythroid, and lymphoid progenitors (Costa et al., 2012; Lin et al., 2014). Several studies, including lineage tracing (Zovein et al., 2008) and in vivo imaging (Boisset et al., 2010) studies, have revealed the endothelial origin of HSCs emerging from a hemogenic endothelium (HE) population within the AGM region. Similarly, earlier waves of hematopoietic progenitors were also shown to derive from the HE (Ema et al., 2006; Lancrin et al., 2010; Nishikawa et al., 1998). The in vitro differentiation of ESCs has been widely used as a model system to dissect and understand the early events of hematopoietic specification in terms of both molecular mechanisms and cellular steps. The careful dissection of this in vitro program has demonstrated that, similarly to in vivo development, blood cells are generated from mesodermal hemangioblast precursors through an HE intermediate (Choi et al., 1998, 2012; Eilken et al., 2009; Fehling et al., 2003; Huber et al., 2004; Kennedy et al., 2007; Lancrin et al., 2009; Wang et al., 2004) and that the same network of transcription factors orchestrates both in vivo and in vitro processes (Moignard et al., 2013). Detailed studies of the generation of primitive erythroid, myeloid, and lymphoid progenitors have suggested a temporal emergence of these blood lineages in vitro, reflecting their sequential emergence in vivo during embryonic development (Irion et al., 2010). This led to the concept that repopulating activity might emerge at late stages of the hematopoietic program during ESC differentiation (Kardel and Eaves, 2012; Lis et al., 2013; Sturgeon et al., 2013) and that the emergence of lymphoid potential marks the establishment of the definitive program (Kennedy et al., 2012; Slukvin, 2013). To date, however, attempts to derive in vivo repopulating hematopoietic cells from late stages of ESC differentiation have been largely unsuccessful. To revisit this long-standing challenge, we took an alternative approach and explored the very first step of hematopoietic specification from the mesoderm. We hypothesized that multilineage progenitors with in vivo repopulating ability might be specified very early upon commitment of mesoderm to the blood program, and might be difficult to maintain as such in the presence of serum or hematopoietic cytokines. We first evaluated the growth factor requirement for optimal specification of hemangioblast to HE. Next, defining the full hematopoietic potential of this emerging population, we observed the concomitant emergence of erythroid, myeloid, and lymphoid progenitors. Interestingly, this early population was also endowed with the capability to engraft immunocompromised mice and to confer multilineage, long-term engraftment. Further studies allowed us to define the temporal emergence of this repopulating ability and to determine the growth factor requirement and immunophenotypic characteristic of this population. Collectively, our findings demonstrate that in vitro repopulating cells emerge very rapidly from mesoderm precursors, are extremely transient, and are exquisitely sensitive to the growth factors present in the differentiating conditions.