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RNA sequencing analysis of 5- and 7-day old salivary gland organoids

ABSTRACT: The specific effects of different radiation types, such as photons and protons, on normal stem cell activity and tissue regeneration remain poorly understood. In our previous work, we characterized salivary gland organoids at 2 days post-irradiation (corresponding to 7-day-old organoids) using single-cell RNA sequencing. To compare the cellular composition of 5-day-old organoids (day of irradiation) with that of 7-day-old organoids, we performed bulk RNA sequencing and applied the deconvolution tool CIBERSORTx, using the 7-day single-cell dataset as a reference. This analysis confirmed that the cell populations in 5- and 7-day-old salivary gland organoids were comparable.

ORGANISM(S): Mus musculus

PROVIDER: GSE303285 | GEO | 2025/09/23

REPOSITORIES: GEO

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Project description:Radiotherapy of head and neck cancer is accompanied by unavoidable co-irradiation of the salivary gland, often resulting in hyposalivation and reduced quality of life. Irradiated salivary gland tissue is characterized by the presence of cellular senescence, fibrosis, and a chronic inflammatory state changing the cellular microenvironment. These microenvironmental changes may be unfavorable for the regenerative potential of tissue stem cells inhibiting regenerative medicine options. Therefore, we investigated microenvironmental changes of the irradiated murine salivary gland and their effect on the potency of salivary gland stem/progenitor cells (SGSCs). After 15 Gy local irradiation of murine salivary glands, alongside an in time advancing decline in function, the submandibular gland (SMG) showed extensive fibrosis, p21-expressing senescent cells, and immune cell infiltration, all indicative of microenvironmental changes. Additionally, irradiated SMG contained a lower number of CD24++/CD29++ and CD24++/CD29+ stem/progenitor cell enrichment marker-expressing cells 30 days post-irradiation which coincides with a reduction in salisphere formation potential over time. However, after subsequent passaging of organoids derived from glands obtained 14 days after irradiation, and to a lesser extent at 30 days post-irradiation, the ability to form secondary organoids recovered to control levels. In contrast, cells obtained from glands at 90, 180, and 360 days post-irradiation irreversibly lost their ability to form organoids, indicating a time-dependent loss of regenerative potential. Transcriptomic analysis showed overexpression of Bmp4 in day 90 organoids derived from irradiated glands compared to the ones from day 14. Inhibition of Bmp4 using Noggin reversed this adverse response suggesting an important role in modulating SGSC function. Thus, early after irradiation, the regenerative potential of surviving SGSCs is preserved and comparable to that of unirradiated cells. At later time points after irradiation, SGSCs lose their regenerative potential suggesting that the prolonged exposure to a deleterious microenvironment leads to an irreversible decline in stemness. Modulation of Bmp4 signaling pathway might improve the regenerative potential of surviving SGSCs and enhance the efficiency of stem cell therapy in restoring the functionality of irradiated salivary glands.

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RNA sequencing analysis of 5- and 7-day old salivary gland organoids

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