IADR Abstract Archives
Functional Epithelial Organoids Induce Salivary Gland Regeneration via Their Exosomes
Objectives: Replacing the secretory units in the hypofunctional salivary gland (SG) damaged by radiotherapy for head and neck cancers is a clinical prerequisite. Here, SG-like epithelial organoids were developed using magnetic 3D bioprinting (M3DB), and their secretome (exosomes) was investigated for SG regeneration after radiotherapy.
Methods: SGo from human dental pulp stem cells (hDPSC) were developed using M3DB in serum-free conditions with different concentrations of FGF10 (4-400ng/ml), an essential SG epithelial signaling cue. Exosomes were isolated from SGo conditioned media by sequential centrifugation and then characterized with transmission electron microscopy, nanoparticle tracking analysis, western blot and mass spectrometry. Afterwards, exosomes were supplemented to irradiated SG ex vivo models to determine epithelial regenerative potential and compare it with the transplanted SGo. Quantitative PCR, whole-mount immunohistochemistry, functional studies for calcium influx and amylase were performed. Data was statistically analyzed by one-way analysis of variance with Tukey posthoc test with a set alpha at 0.05 using GraphPad Prism 8 software.
Results: SGo supplemented with 400ng/ml FGF10 displayed several functional features including calcium influx upon neurostimulation (carbachol and isoproterenol) and amylase production. Exosomes from SGo were abundant and possessed a mode diameter of 79.78 (±2.20) nm and displayed exosome specific markers and FGF signaling cues (including FGF2, FGF7 and FGF10) and downstream effectors. After transplantation, epithelial growth index was significantly increased with SGo exosomes treated glands in both non-irradiated and irradiated SG via an increase in Ki67, Sox2 and β3-tubulin.
Conclusions: SGo can secrete relevant exosomes with the ability to promote epithelial growth during SG homeostasis, and can also regenerate the epithelial compartment in the irradiated SG by increasing cellular mitosis, stem cell populations depleted by radiotherapy and neuronal sprouting. These regenerative effects were mediated by several FGF downstream signaling cues.
Methods: SGo from human dental pulp stem cells (hDPSC) were developed using M3DB in serum-free conditions with different concentrations of FGF10 (4-400ng/ml), an essential SG epithelial signaling cue. Exosomes were isolated from SGo conditioned media by sequential centrifugation and then characterized with transmission electron microscopy, nanoparticle tracking analysis, western blot and mass spectrometry. Afterwards, exosomes were supplemented to irradiated SG ex vivo models to determine epithelial regenerative potential and compare it with the transplanted SGo. Quantitative PCR, whole-mount immunohistochemistry, functional studies for calcium influx and amylase were performed. Data was statistically analyzed by one-way analysis of variance with Tukey posthoc test with a set alpha at 0.05 using GraphPad Prism 8 software.
Results: SGo supplemented with 400ng/ml FGF10 displayed several functional features including calcium influx upon neurostimulation (carbachol and isoproterenol) and amylase production. Exosomes from SGo were abundant and possessed a mode diameter of 79.78 (±2.20) nm and displayed exosome specific markers and FGF signaling cues (including FGF2, FGF7 and FGF10) and downstream effectors. After transplantation, epithelial growth index was significantly increased with SGo exosomes treated glands in both non-irradiated and irradiated SG via an increase in Ki67, Sox2 and β3-tubulin.
Conclusions: SGo can secrete relevant exosomes with the ability to promote epithelial growth during SG homeostasis, and can also regenerate the epithelial compartment in the irradiated SG by increasing cellular mitosis, stem cell populations depleted by radiotherapy and neuronal sprouting. These regenerative effects were mediated by several FGF downstream signaling cues.