The use of a novel Pax

The use of a novel Pax9Venus reporter PSC line allowed the purification of PSC-derived Pax9+ AFE for molecular analysis and enabled the comparison to embryonic Pax9+ AFE. The expression of several pharyngeal endoderm transcription factors in PSC-derived Pax9+ AFE suggests that these Cy3.5 hydrazide cost may have the capacity to differentiate into pharyngeal foregut derivatives. Interestingly, expression levels of several pharyngeal foregut-specific transcription factors were higher in the embryonic Pax9+ AFE than in PSC-derived Pax9+ AFE. It is therefore possible that in vitro Pax9+ AFE represents a more immature cell population and yet to be identified signals are required to specify pharyngeal endoderm derived organ domains. Although we included BMPi and TGFβi in our directed differentiation protocol, consistent with the published PAX9+ AFE generating protocol, our data suggests that these inhibitors may not be required to specify Pax9+ AFE from DE for mouse PSCs. In concordance, other directed differentiation protocols did not rely on exogenous BMP inhibition (Mou et al., 2012) or TGFβ inhibition (Wong et al., 2012) to generate FOXA2+ SOX2+ AFE cells from PSCs. Thus, further work is needed to determine the context in which BMP and TGFβ inhibition are necessary for the generation of functional Pax9+ AFE.
Conclusions We utilized small molecules and chemically defined media to generate PAX9+ AFE cells from mouse and human PSCs. Through the use of a novel reporter Pax9Venus mouse PSC line, we isolated PSC-derived PAX9+EPCAM+ cells that had a gene expression signature consistent with Pax9+ AFE. Upon transplantation in vivo, these cells formed a variety of organized epithelial structures including Pax9+ stratified squamous epithelium, pseudostratified columnar epithelium and mucosal glands. We were able to modify the differentiation protocol for the human stem cell line HUES64, thereby enabling future work on human disease modeling and cell replacement therapies. The following are the supplementary data related to this article.
Acknowledgments We thank S. Morrison, R. Sherwood and A. McMahon for providing the Sox17GFP, p2lox40, and AV3 mouse ESC lines, respectively. We thank L. Atehortua, J. Feng, G. Kenty, L. Paquin, and L. Yagasaki for technical assistance. We thank the UMass FACS core, confocal core and transgenic animal modeling core, DERC morphology core and D. Garlick for assistance with experiments and analysis. We also thank D. Cohen for critical reading of the manuscript and R. Sherwood and J. Rajagopal for helpful discussions. We thank D. Melton for initial financial and logistical support, and for continuing discussions. This work was supported by The Leona M. and Harry B. Helmsley Charitable Trust.
Introduction Neural stem cells (NSCs) in the mammalian brain are characterized by their ability to self-renew and differentiate into multiple types of neuron and glia. They form free floating spheres if cultured in a medium containing growth factors (Fischer et al., 2011). These properties make them much coveted tools in the field of regenerative medicine where it is hoped that they can be utilized to regenerate neurons that have been lost as a result of disease or injury (Ruff and Fehlings, 2010). NSCs are most abundant in the developing fetal brain and persist throughout the whole life generating new neurons and glia continuously. The study of NSCs from adult brain tissue is particularly challenging because the cells are low in number (0.1–1%) and require specialized and tedious cell preparation techniques which have limited yield (Brewer and Torricelli, 2007; Galli et al., 2003). As such, considerable research has been focused on developing isolation techniques for these elusive cells. Major obstacles to isolate NSCs have been the lack of NSC specific cell surface markers and tools to detect intracellular markers which are more specific both in the developing and mature brain. Commonly used methods of NSC labeling either involve antibody for cell surface marker labeling or fluorescent protein expression with the desired marker of interest (Fischer et al., 2011; Corti et al., 2007; Coskun et al., 2008). Presently, the most widely used cell surface markers for the isolation of NSCs are CD133 and SSEA-1 (Capela and Temple, 2002; Corti et al., 2007). The fluorescent dye, AldeFluor which is catalyzed by the intracellular metabolic enzyme aldehyde dehydrogenase to be retained in the cell has also been demonstrated to enrich for NSCs (Corti et al., 2006). More recently, the stem cell specific fluorescent dye CDy1, developed by us as a pluripotent stem cell probe (Im et al., 2010) has also been shown to be able to isolate NSC from the mouse brain although the mechanism of action remains to be elucidated (Vukovic et al., 2013). Using a combination of the three NSC markers (CD133, SSEA-1 and AldeFluor), Obermair et al. reported several subpopulations of neural stem/progenitors within the brain (Obermair et al., 2010). Fischer et al. reported the isolation of NSCs from GFAP+/CD133+ cells from the adult mouse brain (Fischer et al., 2011). On the other hand, other studies have shown that the expression of CD133 in NSC depends on developmental stage (Pfenninger et al., 2007) and that a significant portion of NSC does not express this marker (Sun et al., 2009).