Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5⁺) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5⁺ or Lgr6⁺ cells from taste tissue can generate continuously expanding 3D structures (“organoids”). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2’-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5⁺ cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6⁺ cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5⁺ or Lgr6⁺ cells, validating the use of this model for the study of taste cell generation.Taste bud cells are heterogeneous and undergo constant turnover (1); however, the origins and generation of taste buds in adult mammals remain largely unclear. Based on morphological and functional characteristics, there are at least three different types of mature taste bud cells [type 1 (glial-like cells), type 2 (receptor cells, including those responsible for sensing sweet, bitter, and umami stimuli), and type 3 (presynaptic cells, including sour sensors)], and well as one type of immature taste bud cell [type 4 (basal cells that are precursors of other types of mature taste cells)] (2, 3). Mature taste bud cells are postmitotic and short-lived, with average life spans estimated at 8–12 d (4, 5), although distinct subtypes of taste bud cells may have different life spans (1, 4, 5). At present, the stem cell population and the regenerative process from adult taste stem/progenitor cells to mature taste bud cells are not well characterized.Lgr5 (leucine-rich repeat-containing G protein-coupled receptor 5), encoded by a Wnt (wingless-type MMTV integration site family) target gene, marks adult stem/progenitor cells in taste tissue in posterior tongue that in vivo give rise to all major types of taste bud cells, as well as perigemmal cells (6, 7). Lgr5 is also known to mark actively cycling stem cells in small intestine, colon, stomach, and hair follicle, as well as quiescent stem cells in liver, pancreas, and cochlea (8). Isolated Lgr5⁺ adult stem cells from multiple tissues are able to generate so-called organoid structures ex vivo (9–11). For instance, Sato and colleagues (10) developed a 3D culture system to grow crypt-villus organoids from single intestinal stem cells; all differentiated cell types were found in these structures, indicating the multipotent nature of these cells. We hypothesized that Lgr5⁺ taste stem/progenitor cells in a 3D culture system would be capable of expanding and giving rise to taste receptor cells ex vivo. In the present study, we isolated Lgr5⁺ stem/progenitor cells from taste tissue and cultured them in a 3D culture system. Single Lgr5⁺ cells grew into organoid structures ex vivo in defined culture conditions, with the presence of both proliferating cells and differentiated mature taste cells in which taste signaling components are functionally expressed. When organoids were replated onto a 2D surface precoated with laminin and polylysine, cells grew out of the organoids and attached to the flat surface, and some cells retained the expressed taste signaling elements and responded to taste stimuli.Lgr5 marks adult taste stem/progenitor cells in posterior tongue, which was shown using an engineered mouse model in which enhanced green fluorescent protein (EGFP) and tamoxifen-inducible Cre recombinase (CreERT2) are knocked-in to replace the coding sequence of Lgr5 and act as surrogate markers for Lgr5 (6, 7). Although Lgr5 is present in fungiform papillae in anterior tongue during embryonic stages and early life, based on the intrinsic GFP signal from the Lgr5-EGFP transgene, Lgr5-EGFP signal could not be detected in fungiform papillae cells in adult mice (6, 7). Therefore, taste stem/progenitor cells remain to be identified in fungiform papillae in anterior tongue. We hypothesized that Lgr6, an Lgr5 homolog, may mark adult taste stem/progenitor cells in anterior tongue, prompted by the finding that Lgr6 is preferentially expressed in taste tissue, but not in the surrounding epithelium devoid of taste tissue (12). Using the Lgr6-EGFP-ires-CreERT2 mouse line (13), we here show that Lgr6 is expressed in cells at the basal area of taste buds in fungiform and circumvallate papillae. By genetic lineage tracing, we show that Lgr6⁺ cells give rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR shows that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Similar to Lgr5⁺ cells, isolated Lgr6⁺ cells can build taste organoids that generate mature taste cells.