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Single Lgr5- Or Lgr6-Expressing Taste Stem/Progenitor Cells Generate Taste Bud Cells Ex Vivo

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Single Lgr5- Or Lgr6-Expressing Taste Stem/Progenitor Cells Generate Taste Bud Cells Ex Vivo Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo Wenwen Rena, Brian C. Lewandowskia, Jaime Watsona, Eitaro Aiharab, Ken Iwatsukic, Alexander A. Bachmanova, Robert F. Margolskeea, and Peihua Jianga,1 aMonell Chemical Senses Center, Philadelphia, PA 19104; bDepartment of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH 45267; and cDepartment of Nutritional Science and Food Safety, Faculty of Applied Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 8, 2014 (received for review May 19, 2014) Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) organoids from single intestinal stem cells; all differentiated cell and its homologs (e.g., Lgr6) mark adult stem cells in multiple types were found in these structures, indicating the multipotent + tissues. Recently, we and others have shown that Lgr5 marks adult nature of these cells. We hypothesized that Lgr5 taste stem/pro- taste stem/progenitor cells in posterior tongue. However, the genitor cells in a 3D culture system would be capable of expanding + regenerative potential of Lgr5-expressing (Lgr5 ) cells and the and giving rise to taste receptor cells ex vivo. In the present study, + identity of adult taste stem/progenitor cells that regenerate taste we isolated Lgr5 stem/progenitor cells from taste tissue and cul- + tissue in anterior tongue remain elusive. In the present work, we tured them in a 3D culture system. Single Lgr5 cells grew into + + describe a culture system in which single isolated Lgr5 or Lgr6 organoid structures ex vivo in defined culture conditions, with the cells from taste tissue can generate continuously expanding 3D presence of both proliferating cells and differentiated mature taste structures (“organoids”). Many cells within these taste organoids cells in which taste signaling components are functionally were cycling and positive for proliferative cell markers, cytokeratin expressed. When organoids were replated onto a 2D surface pre- K5 and Sox2, and incorporated 5-bromo-2’-deoxyuridine. Impor- coated with laminin and polylysine, cells grew out of the organ- tantly, mature taste receptor cells that express gustducin, carbonic oids and attached to the flat surface, and some cells retained the anhydrase 4, taste receptor type 1 member 3, nucleoside triphos- expressed taste signaling elements and responded to taste stimuli. phate diphosphohydrolase-2, or cytokeratin K8 were present in Lgr5 marks adult taste stem/progenitor cells in posterior the taste organoids. Using calcium imaging assays, we found that + tongue, which was shown using an engineered mouse model in cells grown out from taste organoids derived from isolated Lgr5 which enhanced green fluorescent protein (EGFP) and tamoxi- cells were functional and responded to tastants in a dose-depen- + fen-inducible Cre recombinase (CreERT2) are knocked-in to dent manner. Genetic lineage tracing showed that Lgr6 cells gave replace the coding sequence of Lgr5 and act as surrogate rise to taste bud cells in taste papillae in both anterior and poste- markers for Lgr5 (6, 7). Although Lgr5 is present in fungiform rior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may papillae in anterior tongue during embryonic stages and early mark the same subset of taste stem/progenitor cells both anteri- life, based on the intrinsic GFP signal from the Lgr5-EGFP BIOLOGY orly and posteriorly. Together, our data demonstrate that func- + transgene, Lgr5-EGFP signal could not be detected in fungiform DEVELOPMENTAL tional taste cells can be generated ex vivo from single Lgr5 or + papillae cells in adult mice (6, 7). Therefore, taste stem/pro- Lgr6 cells, validating the use of this model for the study of taste genitor cells remain to be identified in fungiform papillae in cell generation. anterior tongue. We hypothesized that Lgr6, an Lgr5 homolog, Lgr5 | Lgr6 | taste stem cells | taste progenitor cells 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 aste bud cells are heterogeneous and undergo constant turnover (1); however, the origins and generation of taste T Significance buds in adult mammals remain largely unclear. Based on mor- phological and functional characteristics, there are at least three different types of mature taste bud cells [type 1 (glial-like cells), Taste tissue regenerates continuously throughout the life span type 2 (receptor cells, including those responsible for sensing in mammals. Here, using lineage tracing and a culture system, sweet, bitter, and umami stimuli), and type 3 (presynaptic cells, we show that leucine-rich repeat-containing G protein-coupled including sour sensors)], and well as one type of immature taste receptor 5-expressing and leucine-rich repeat-containing G bud cell [type 4 (basal cells that are precursors of other types of protein-coupled receptor 6-expressing taste stem/progenitor mature taste cells)] (2, 3). Mature taste bud cells are postmitotic cells generate mature taste cells in vivo and ex vivo. Impor- and short-lived, with average life spans estimated at 8–12 d (4, 5), tantly, our ex vivo studies show that single-progenitor cells can although distinct subtypes of taste bud cells may have different generate all mature taste cell types and that differentiated taste cells form in the absence of innervation. This ex vivo life spans (1, 4, 5). At present, the stem cell population and the model mimics the development of taste bud cells in taste pa- regenerative process from adult taste stem/progenitor cells to pillae, recapitulates the process of taste renewal from adult mature taste bud cells are not well characterized. taste stem cells to mature taste cells, and provides a means to Lgr5 (leucine-rich repeat-containing G protein-coupled re- study the regulation of taste cell generation and to understand ceptor 5), encoded by a Wnt (wingless-type MMTV integration the origins and cell lineage relationships within taste buds. site family) target gene, marks adult stem/progenitor cells in taste tissue in posterior tongue that in vivo give rise to all major Author contributions: W.R., R.F.M., and P.J. designed research; W.R., B.C.L., and J.W. types of taste bud cells, as well as perigemmal cells (6, 7). Lgr5 is performed research; E.A., K.I., and A.A.B. contributed new reagents/analytic tools; W.R., also known to mark actively cycling stem cells in small intestine, B.C.L., R.F.M., and P.J. analyzed data; and R.F.M. and P.J. wrote the paper. colon, stomach, and hair follicle, as well as quiescent stem cells The authors declare no conflict of interest. + in liver, pancreas, and cochlea (8). Isolated Lgr5 adult This article is a PNAS Direct Submission. stem cells from multiple tissues are able to generate so-called 1To whom correspondence should be addressed. Email: [email protected]. – organoid structures ex vivo (9 11). For instance, Sato and col- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. leagues (10) developed a 3D culture system to grow crypt-villus 1073/pnas.1409064111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1409064111 PNAS | November 18, 2014 | vol. 111 | no. 46 | 16401–16406 Downloaded by guest on October 1, 2021 tissue (12). Using the Lgr6-EGFP-ires-CreERT2 mouse line (13), organoids showed no detectable EGFP signal; the EGFP signal we here show that Lgr6 is expressed in cells at the basal area of decreased rapidly and became undetectable in 2–3 d. However, taste buds in fungiform and circumvallate papillae. By genetic organoids were occasionally found to retain a strong EGFP sig- + lineage tracing, we show that Lgr6 cells give rise to taste bud nal: in two of 12 preparations, organoids with GFP fluorescence cells in taste papillae in both anterior and posterior tongue. RT- were seen in 43 (12%) of 374 organoids and 61 (5%) of 1201 PCR shows that Lgr5 and Lgr6 may mark the same subset of organoids examined. Presumably this continued expression of GFP-fluorescence resulted from Lgr5-EGFP cells that were ex- taste stem/progenitor cells both anteriorly and posteriorly. Sim- + + + panded from single isolated Lgr5-EGFP cells (Fig. 1E, Bottom). ilar to Lgr5 cells, isolated Lgr6 cells can build taste organoids + To assess the clonality of isolated Lgr5 cells and to determine that generate mature taste cells. + whether organoids truly grow out from single Lgr5 cells, as Results opposed to small aggregates of cells, we crossed Lgr5-EGFP-ires- + CreERT2 mice with Rosa26-tdTomato mice to generate Lgr5- Single Isolated Lgr5 Cells Generate Taste Organoids. To determine + − + − + / / whether Lgr5 taste stem/progenitor cells are capable of EGFP-ires-CreERT2 ; Rosa26-tdTomato mice in which tamoxifen-induced Cre generates expression of tdTomato expanding and generating taste cells in vitro, and to establish + + fluorescence protein (red) to mark cells from the Lgr5 lineage. a taste culture system, we purified Lgr5 taste stem/progenitor +/− +/− cells (Fig. 1A) from Lgr5-EGFP-ires-CreERT2 mice, using fluo- Half the Lgr5-EGFP-ires-CreERT2 ; Rosa26-tdTomato mice were injected with tamoxifen 1 d before cell sorting to mark rescence-activated cell sorting (FACS), based on the green + + Lgr5 progeny, using tdTomato fluorescence. Then we purified fluorescence signal of Lgr5-EGFP cells (Fig. 1 B and C). The cells that were positive for EGFP (green) via FACS from both cell sorting gates for isolating GFP-expressing cells were set such tamoxifen-treated and untreated mice and cultured them in that no cells from wild-type littermate controls were isolated mixture. Using this strategy, tamoxifen-induced Cre generated (Fig. 1B). All sorted cells expressed EGFP, as demonstrated by + expression of tdTomato fluorescence protein (red) in only a + the green fluorescence signal (Fig.
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