Murine circumvallate papilla taste bud organoid development and characterization of stem/progenitor cell cycle

There are limited models to study taste function, and here we generate taste bud‐like structures (“organoids”) from native circumvallate papilla tissue as well as single flow‐sorted Lgr5 + cells that develop structural and phenotypic characteristics similar to native tissue. Organoids have a multilayered epithelium with CD44+ and SOX9+ stem/progenitor in the outer layers and taste receptor‐expressing and gustducin‐expressing taste cells in the inner layers. 3D time lapse monitoring of taste bud organoids created from cell cycle reporter mice (FUCCI2 transgene G1: mVenus, S/G2/M: mCherry) or H2B‐GFP transgenic mice, shows that stem/progenitor cells migrate to form taste bud‐like structures and have cyclical 24 hrs of peaks of proliferation. During tracking of individual cells, however, a diversity of several cell cycle durations, were noted; 12.5 ± 0.3, 22.2 ± 0.7, 31.8 ± 0.6, 42.0 ± 0.9, and >50 hr, in 41.2, 23.5, 20.6, 8.8, and 2.9 percent of total analyzed cells, respectively. In organoids with cell numbers <400, 42% daughter cells had shorter cell cycle duration than their parent cells. In organoids with cell numbers ranging between 400‐800, 40% of daughter cells mimicked their parent cell cycle time. In organoids >800 cells, parental cells tended to have longer cell cycle than daughter cells. In conclusion, we resolve multiple distinct populations of replicating cells in taste bud development, and suggest that spatial and cell confluence feedback mechanisms impact progenitor cell growth. A population of rapidly cycling cells have the capacity to migrate up the taste bud niche, with implications on the mechanisms of homeostatic turnover of taste cells.