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Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells.

ABSTRACT: The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5^{EGFP-IRES-CreERT2} allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

SUBMITTER: Shechtman LA

PROVIDER: S-EPMC8336305 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:On the basis of their self-renewal capacity and their ability to differentiate into derivatives of all three germ layers, germ line-derived multipotent adult stem cells (maGSCs) from mouse testis might serve as one of preferable sources for pluripotent stem cells in regenerative medicine. In our study, we aimed for an efficient hepatic differentiation protocol that is applicable for both maGSCs and embryonic stem cells (ESCs). We attempted to accomplish this goal by using a new established co-culture system with OP9 stroma cells for direct differentiation of maGSCs and ESCs into hepatic cells. We found that the hepatic differentiation of maGSCs was induced by the OP9 co-culture system in comparison to the gelatin culture. Furthermore, we showed that the combination of OP9 co-culture with activin A resulted in the increased expression of endodermal and early hepatic markers Gata4, Sox17, Foxa2, Hnf4, Afp, and Ttr compared to differentiated cells on gelatin or on OP9 alone. Moreover, the hepatic progenitors were capable of differentiating further into mature hepatic cells, demonstrated by the expression of liver-specific markers Aat, Alb, Tdo2, Krt18, Krt8, Krt19, Cps1, Sek, Cyp7a1, Otc, and Pah. A high percentage of maGSC-derived hepatic progenitors (51% AFP- and 61% DLK1-positive) and mature hepatic-like cells (26% ALB-positive) were achieved using this OP9 co-culture system. These generated hepatic cells successfully demonstrated in vitro functions associated with mature hepatocytes, including albumin and urea secretion, glycogen storage, and uptake of low-density lipoprotein. The established co-culture system for maGSCs into functional hepatic cells might serve as a suitable model to delineate the differentiation process for the generation of high numbers of mature hepatocytes in humans without genetic manipulations and make germ line-derived stem cells a potential autologous and alternative cell source for hepatic transplants in metabolic liver disorders.

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Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells.

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