Organoid Modeling of Mouse Anterior Tongue Epithelium Reveals Regional and Cellular Identities

The tongue is essential for swallowing, taste perception, and mechanosensation. The anterior and posterior parts of the tongue have region-specific developmental origins and are maintained by adult epithelial stem/progenitor cells. In vitro models that can be used to investigate anterior tongue biology have been lacking. Here, we develop a protocol to generate a long-term expanding organoid model from adult mouse dorsal anterior tongue. Anterior tongue organoids consist of Lgr6+ cells, Sox2+ stem/progenitor cells, and Hoxc13+ filiform papillae progenitor cells. Furthermore, anterior tongue organoids share region-specific transcriptomic profiles, gene regulatory networks, and signaling pathways with anterior tongue tissue. Anterior tongue organoids can be differentiated into various epithelial cell types including Merkel-like cells, keratinocytes, and taste bud cells. Gene regulatory network analysis reveals transcriptional programs associated with Krt8+ cell and Krt23+/Sbsn+ keratinocyte differentiation in the organoids. Together, this study provides an in vitro model of mouse dorsal tongue epithelium. Overall design: Tongues were collected from 20 BL/6 mice. CvP and FoP were collected using tweezers under stereomicroscope. Tissues were minced and treated with TrypLE containing 10 µM Y-27538 for up to 30 minutes. During the enzymatic digestion process, every 5 minutes, tissues were pipetted several times using a 1000 uL pipette tip. Single cells and small tissue clumps were collected in cold medium and fresh TrypLE + 10 uM Y-27538 was added to further digest the remaining tissues. In brief, 300 – 400 µL of 1 mg/mL collagenase/dispase solution was injected under the lingual epithelium using a 27G needle and tongues were incubated at 37°C for 20 to 25 minutes. Then, the epithelium was peeled off and digested with TrypLE containing 10 µM Y-27538 in the same manner as CvP and FoP tissues. ATEM and ATDM were collected and processed in the similar manner as the tissues. Single cells were washed twice with cold FACS buffer containing 1% FBS, 0.1 mM EDTA, and PBS. Cell pellets were resuspended in 500 µL cold FACS buffer, filtered through a 70 µm cell strainer and then through a 40 µm FACS tube. Cells were stained with 5 µM Draq5 (424101; Biolegend) and 1 µM 4',6-diamidino-2-phenylindole (DAPI, D9542; Sigma-Aldrich) according to the manufacturer's protocols. Viable single cells (Draq5+/DAPI-) were collected in PBS containing 1% BSA and 10 µM Y-27538 using the SH800S Cell Sorter (Sony Biotechnology, CA, USA). Cell numbers were counted using a hemocytometer before and after cells were washed with PBS containing 1% BSA and 10 µM Y-27538. ATDM: Anterior tongue organoids in differentiation medium. ATEM: Anterior tongue organoids in expansion medium. PTEM: Posterior tongue organoids in expansion medium.