One approach to generate specific cell populations
One approach to generate specific cell populations is to direct PSCs toward a desired cell type by sequential progression through intermediate cell populations, mimicking the stages of embryonic development. Monolayer differentiation approaches allow homogeneous accessibility of GS-9973 to exogenous signals required to guide cell fate decisions. Indeed, monolayer differentiation strategies have been employed to produce definitive endoderm (DE), the precursor of AFE, from PSCs through activation of the wingless (WNT) and TGFβ signaling pathways (Sherwood et al., 2011; D\'Amour et al., 2005). More recently, AFE has been generated from PSC-derived definitive endoderm (DE) in vitro and upon further differentiation can be directed to express the lung- and thyroid-specific transcription factor Nkx2.1 (Mou et al., 2012; Longmire et al., 2012; Wong et al., 2012; Green et al., 2011). The generation of organized AFE epithelia has also been reported, however these are currently limited to respiratory epithelial lineages (Mou et al., 2012; Wong et al., 2012), necessitating further work to produce additional organized AFE epithelia from PSCs. In order to develop a protocol to produce AFE capable of forming a broader spectrum of AFE-derived epithelia, we selected the transcription factor Pax9 for use as an AFE marker based on its broad expression domain (Fig. 1A). Pax9 is expressed from e9.0 in the developing murine pharyngeal foregut endoderm and is required for the development of the thymus, parathyroid and ultimobranchial bodies (Peters et al., 1998). Pax9 expression is retained in several AFE-derived mature cell types including the oral epithelium, salivary glands, parathyroid gland, thymus, esophagus and fore-stomach (Peters et al., 1998). Although the generation of PAX9+ cell containing AFE cultures has been reported from human PSCs (Green et al., 2011), upon transplantation, obtained organized epithelial structures expressed genes indicative of lung epithelial cells rather than other PAX9+ AFE derived epithelia. Thus, there is a need to explore alternative approaches to generate PAX9+ cells from PSCs in order to produce the spectrum of PAX9+ AFE epithelia.
We report a monolayer differentiation approach using small molecules and chemically defined media to generate PAX9+ AFE cells from both mouse and human PSCs, thereby minimizing the contribution of uncharacterized signals during directed differentiation. Through the use of a novel mouse reporter PSC line, we isolated Pax9+ AFE cells for molecular and functional characterization. Generated Pax9+ AFE cells not only exhibit molecular signatures of anterior and pharyngeal foregut in vitro, but also are competent to form a variety of organized AFE epithelial structures upon transplantation in vivo. Thus, these results advance the AFE differentiation field by generating a transcriptional signature for Pax9+ AFE, broadening the spectrum of AFE epithelia currently generated from PSCs, and creating a platform to study AFE developmental disorders.
Materials and Methods
Discussion The generation of specific AFE-derived cells allow for novel cell therapies and human disease models for diseases affecting organs originating from the anterior foregut endoderm. Here, we report a defined media and small molecule-based differentiation protocol to produce Pax9+ AFE from PSCs that when matured in vivo are competent to organize into several distinct types of anterior foregut epithelia. This generation of a small-molecule directed, defined protocol will be beneficial for future production of cells for replacement therapies, and to advance the understanding of the essential signals required for differentiation. This protocol presents a significant advance over previous protocols designed to generate PAX9+ AFE, as the AFE cells generated in this report have the potential to give rise to multiple AFE-derived organized PAX9+ epithelial structures including stratified squamous epithelium, pseudostratified columnar epithelium and mucosal glands.