Development of human taste organoid models

The sense of taste is mediated by taste buds on the tongue. Each bud is a heterogeneous collection of taste receptor cells (TRCs), and all TRCs, which transduce sweet, bitter, salt, sour and umami tastes, are continually renewed from adult stem cells outside of taste buds. However, taste function can be perturbed by numerous

insults, including drugs and viral infection that affect TRC renewal. Ex vivo organoids are now a conventional method for study of cellular and molecular mechanisms of adult epithelial homeostasis, e.g., intestine. We and others have recently established this technology for the taste system, where isolated stem cells from the circumvallate taste papilla (CVP) of adult mice are cultured to

generate TRC-containing lingual organoids; this model is used increasingly to study the impact of drugs and pathogens on taste homeostasis. While informative, however, results in mice may not precisely reflect how taste is perturbed in patients. Therefore, here we proposed to establish human TRC-replete lingual organoid

lines from CVPs obtained postmortem from organ donors as a first step in developing human in vitro models. Our strategy overcomes several logistical and technical barriers to production of lingual organoids in humans. 1. We now have a pipeline to obtain CVP biopsies through a non-profit organ and tissue procurement

organization. 2. We are expert at mouse lingual organoids and will apply our skill to human tissue. 3. We will work closely with our Organoid and Tissue Modelling Core, who have already created organoid lines from human postmortem donor pancreas and intestine. Aim 1. Validate and characterize CVP samples in male and female human donor tissue. Humans have

multiple, easily visible CVPs. Initially, PSR staff will process harvested biopsies to verify CVPs are reliably obtained. Subsequently, TRC and stem cell marker gene expression will be assessed via q-PCR, in situ hybridization (ISH) and/or immunofluorescence (IF). We will perform single cell RNA-sequencing (scRNA-

seq) of fresh male and female biopsies to transcriptionally profile CVP epithelial cell populations, identify differentially expressed genes in human TRCs and stem cells, and construct lineage models. Aim 2. Establish human lingual organoids from CVP biopsies. Initially, small tissue pieces of CVPs will be

cultured under our mouse lingual organoid protocol. Organoids will be assessed for TRC and stem cell marker gene expression via qPCR, ISH and IF as in Aim 1. The organoid protocol will be modified as necessary; permutations may include differences in treatment of starting material, plating density, culture media

components, and duration of culture. Once TRC-replete organoids are produced reliably, organoid lines from at least 2 female and 2 male donors will be maintained through serial passage and storage of frozen stocks. Additionally, we will perform scRNA-seq of 1 male and 1 female organoid line for bioinformatic comparison

with CVP datasets obtained in Aim 1.