During embryonic development retinoic acid

During embryonic development, retinoic concentration equation (RA) acts as a morphogen, providing signals that instruct commitment of unspecified precursors toward separate cell fates, thereby helping to mediate tissue patterning and organogenesis (Duester, 2008; Kumar and Duester, 2011; Ross et al., 2000). As such, RA is also a potent teratogen capable of disturbing developmental processes, causing severe malformations of the fetus. RA is a signaling molecule derived from vitamin A (retinol), regulating cellular proliferation and differentiation, and is produced by cells that express the enzymes retinaldehyde dehydrogenase 1 (RALDH1), RALDH2, or RALDH3. A small molecule, diethylaminobenzaldehyde (DEAB), inhibits the activity of these enzymes and can be used to limit endogenous RA signaling (Moreb et al., 2012; Perz-Edwards et al., 2001; Russo and Hilton, 1988). When available, RA enters the nucleus to bind the retinoic acid receptor (RAR) family of nuclear receptors that, in turn, by forming heterodimeric complexes with the retinoid-X-receptor (RXR) family, localize to specific retinoic acid response elements (RAREs) in promoter regions of the genome to drive transcription of RA target genes. Modulation of RA in in vitro models of development provides a useful tool toward understanding commitment into tissues that depend either on high levels of RA, such as the developing ectodermal and endodermal derivatives (Murry and Keller, 2008) or on low levels of RA such as the posterior patterning of the lateral plate mesoderm (LPM) (Deimling and Drysdale, 2009). While several model organisms including Xenopus (Xenopus laevis), zebrafish (Danio rerio), and mouse (Mus musculus) have been used to study the effects of RA on embryonic development, similar studies have not been possible in the human setting for both technical and ethical reasons. In addition to its role in embryonic development, RA is also known to have strong effects on the hematopoietic lineage. However, the studies on the role of RA in regulating adult hematopoiesis have yielded opposing results. For example, RA signaling inhibition expands hematopoietic progenitors in both the human and murine setting (Chute et al., 2006; Muramoto et al., 2010), while other studies report increased maintenance and self-renewal upon adding RA to short- and long-term repopulating hematopoietic stem cells (HSCs) in the murine setting, mediated through activation of the RARγ receptor (Purton et al., 2000, 2006). These apparent contradictions likely reflect the increasingly complex nature of RA signaling that is emerging in the field, which is why a more contextual understanding might provide a better model to explain these previous findings. Indeed, work analyzing the effects of RA on different subpopulations of adult blood demonstrated that RA has a pleiotropic effect, enhancing the self-renewal of the HSC compartment while accelerating the differentiation of downstream progenitors (Purton et al., 1999). Several groups have created protocols that allow in vitro generation of hematopoietic cells from human pluripotent stem cells (Kardel and Eaves, 2012; Murry and Keller, 2008; Woods et al., 2011), with more recent reports demonstrating the generation of definitive hematopoietic cells capable of differentiation into the lymphoid lineage (Kennedy et al., 2012; Timmermans et al., 2009). However, efficient generation of hematopoietic stem and progenitor cells, with functional properties of their in vivo counterparts, remains challenging, possibly due to the current limitations to how accurately the conditions of embryonic blood development can be reproduced in vitro. In this study, using a human pluripotent stem cell differentiation system, we model hematopoietic development and increase the generation efficiency of hematopoietic cells with an HSC-like phenotype by limiting RA signaling during directed pluripotent stem differentiation toward blood. RA signaling inhibition maintains immature progenitors in a less differentiated state and enables efficient commitment toward the hematopoietic lineage during multiple stages of the developmental process. Our results define the role of RA in hematopoietic differentiation and provide evidence that low levels of RA signaling promote hematopoietic development from pluripotent stem cells.