br Results br Discussion Our data demonstrate that both

Results
Discussion Our data demonstrate that both the emergence and maintenance of in vivo repopulating Exendin-3 (9-39) amide are extremely sensitive to the cytokine milieu: a change in a single factor will completely abolish the detection of engrafting cells. When we compared the outcome of FLK1 cells cultured in the presence of BAFV versus BFV, the absence of Activin A dramatically affected the detection of in vivo engrafting cells, as they were most likely rapidly pushed toward differentiation when exposed only to BFV. Taken together, our results strongly suggest that the signaling pathway activated by Activin A, but not Nodal, is critical, but not sufficient, for maintaining the repopulating ability of the TIE2+cKIT+ population. The addition of FGF seems to reinforce the role of Activin A; however, on its own, FGF is not able to maintain the population of engrafting hematopoietic progenitors. Finally, BMP4 signaling appears to be critical for conferring hematopoietic competency to the HE, an observation that is consistent with the known role of BMP4 in the regulation of Runx1 (Burns et al., 2005; Pimanda et al., 2007). Although our study identifies differentiating conditions that allow the detection of engrafting hematopoietic cells, further work will be required to improve and optimize the culture conditions for the maintenance and expansion of these repopulating cells. An important conceptual aspect of our findings relates to the relationship between ESC-derived repopulating cells and their in vivo counterparts. Based on their immunophenotypic characteristics, the ESC-derived cells more closely resemble the VE-cad+CD45−CD41low pre-HSC type I population identified in the AGM region at E11.5 (Rybtsov et al., 2011). However, these type I pre-HSCs express a low level of CD41 and do not engraft recipients unless they are cocultured for 4 days with OP9, in contrast to ESC-derived repopulating cells, which do not express CD41 and are able to engraft directly, albeit when injected intrafemorally. Based on their limited self-renewal characteristics, the ESC-derived engrafting cells might correspond to lineage-committed progenitors with a repopulating ability in which the multilineage potential is dissociated from the self-renewal capacity, as recently described for adult mouse bone marrow progenitors (Yamamoto et al., 2013). An interesting finding in our study is the concomitant emergence of primitive erythroid, myeloid, definitive erythroid, and lymphoid potential very early upon mesoderm specification. This is in contrast to previous studies that reported the sequential generation of these progenitors during in vitro differentiation of ESCs (Keller et al., 1993; Kennedy et al., 2012; Rafii et al., 2013). In those studies, serum-supplemented factors may have conditioned or altered the timing of differentiation and delayed the emergence of specific progenitor subsets. During embryonic development, the emergence of hematopoietic progenitors occurs in successive waves, with primitive erythroid progenitors emerging first around E7.25, followed by erythro-myeloid progenitors from E8.25 and lymphoid progenitors from E9, whereas definitive HSCs are only detected from E10.5 onward (Costa et al., 2012; Lin et al., 2014). One possible explanation to account for our findings is that during serum-free ESC differentiation, all hematopoietic programs unravel simultaneously because there are no extrinsic factors restricting or altering the developmental timing for the emergence of each lineage. During embryonic development, these cues are provided by the microenvironment in which these precursors reside.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction One key etiological factor underlying a wide range of diseases is the progressive decline in immune function with age (Dorshkind et al., 2009). At its core is a reduction in lymphopoiesis within the bone marrow (BM) and thymus (Miller and Allman, 2003; Rodewald, 1998), attributed in part to a decrease in the number and function of lymphoid progenitors (Min et al., 2004, 2006). Increasing evidence suggests that intrinsic changes to the earliest hematopoietic stem cells (HSCs) also contribute toward age-related immune degeneration (Geiger et al., 2013). Deficiency in DNA repair, altered DNA methylation patterns, aberrant metabolism and reactive oxygen species, and skewed upregulation of myeloid- (at the expense of lymphoid-) associated genes all contribute to altered HSC function with age (expertly reviewed in Geiger et al., 2013). However, in addition to intrinsic functional changes, extrinsic alterations to the HSC niche also likely to contribute toward the degeneration of HSC function with age (Woolthuis et al., 2011).