Generation of organized anterior foregut epithelia from pluripotent stem cells using small molecules

Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1+ lung and thyroid lineages. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9+ AFE cells, which when transplanted in vivo, can form several distinct complex AFE-derived epithelia, including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus, this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders. •PSCs can be differentiated to AFE using small molecules and defined media.•A novel Pax9 mouse reporter cell line allows purification and characterized of AFE.•Gene expression signature characterizes Pax9+AFE as anterior and pharyngeal endoderm.•In vivo maturation allows formation of various organized AFE epithelia from Pax9+ AFE.