Journal Article

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

Bachmanov, Alexander A., Margolskee, Robert F., Jiang, Peihua, Iwatsuki, Ken, Lewandowski, Brian C., Aihara, Eitaro, Ren, Wenwen, Watson, Jaime

2014

Overview

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. Significance Taste tissue regenerates continuously throughout the life span in mammals. Here, using lineage tracing and a culture system, we show that leucine-rich repeat-containing G protein-coupled receptor 5-expressing and leucine-rich repeat-containing G protein-coupled receptor 6-expressing taste stem/progenitor cells generate mature taste cells in vivo and ex vivo. Importantly, our ex vivo studies show that single-progenitor cells can generate all mature taste cell types and that differentiated taste cells form in the absence of innervation. This ex vivo model mimics the development of taste bud cells in taste papillae, recapitulates the process of taste renewal from adult taste stem cells to mature taste cells, and provides a means to study the regulation of taste cell generation and to understand the origins and cell lineage relationships within taste buds.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: adults / Animals / Biological Sciences / Biomarkers / Cell lines / Cell Separation / Cells, Cultured / Citric Acid - pharmacology / Cultured cells / G-protein coupled receptors / Gene expression / Genes, Reporter / Genetics / Glycoproteins / Green Fluorescent Proteins - analysis / Green Fluorescent Proteins - genetics / Gustatory perception / In Vitro Techniques / innervation / longevity / mammals / Mice / Mice, Transgenic / Microscopy, Fluorescence / Multipotent stem cells / Organoids / palps / Quaternary Ammonium Compounds - pharmacology / Receptors / Receptors, G-Protein-Coupled - genetics / Receptors, G-Protein-Coupled - physiology / Recombinant Fusion Proteins - analysis / Recombinant Fusion Proteins - biosynthesis / Sodium Chloride - pharmacology / Sodium Glutamate - pharmacology / Stem cells / Stem Cells - cytology / Sucrose - analogs & derivatives / Sucrose - pharmacology / T cell receptors / Tamoxifen - pharmacology / Taste / Taste - physiology / Taste buds / Taste Buds - cytology / Taste Buds - metabolism / Taste cells / Thiazines - pharmacology / Tissues / Tongue / Tongue - cytology