Salivary glands, which mainly consist of three major glands, namely the parotid gland (PG), submandibular gland (SMG), sublingual gland (SLG), and numerous minor salivary glands, play an indispensable role in the physiological processes of the human body. These glands work together to produce and secrete saliva, which is crucial for multiple physiological functions of the human body, including lubrication, digestion, immunity, and maintaining the balance within the oral cavity.

Organoids are three-dimensional structures formed by self - organization and differentiation of stem cells or progenitor cells in vitro. Their morphology, structure, and function are highly similar to those of in vivo organ tissues, making them a highly promising research model. For oral diseases, due to the particularity of their tissues and locations, the development of appropriate organoid models is undoubtedly the best choice for understanding the pathogenesis and accelerating the drug development process.

In June 2022, Jong-Eun Park & Jae-Yol Lim et al. published an article titled "Salivary gland organoid culture maintains distinct glandular properties of murine and human major salivary glands" in Nature Communication. In this study, the researchers successfully established salivary gland organoids that can be cultured for a long time, including salivary gland acini, ducts, and myoepithelial cells from the major salivary glands of murine and humans.

Materials and Methods

Murine salivary gland tissues were obtained from the parotid gland, submandibular gland, and sublingual gland of 6 - 8-week-old female C57BL/6 mice and were reared under specific conditions. The tissues were processed and embedded in Matrigel and then cultured in the mouse growth and expansion medium (GEM) containing various growth factors and small molecules. Specific factor adjustments were made during differentiation. Human salivary gland tissues were obtained from patients with related diseases with consent. After processing, they were cultured in the human GEM with adjusted components. A variety of detection and analysis methods such as quantitative real-time PCR analysis, organoid proliferation assay, image acquisition (bright field, H&E, PAS, IHC, IF images), functional swelling assay, calcium influx assay, karyotype analysis, whole exome sequencing, flow cytometry analysis and sorting, bulk RNA sequencing, single-cell RNA sequencing, CRTC1 - MAML2 fusion gene detection, and tumor organoid radiation model were used to study the salivary gland tissues and organoids. Each method was operated according to the corresponding standard procedures and reagents.

Results

Firstly, they established a murine salivary gland organoid culture system with GEM. All its factors are crucial for organoid passage/growth (Fig. 1a-b). In DAM, SMG organoids showed more salivary gland-like traits (Fig. 1c-d). They retained gland specificity and proliferation marker expression in GEM and DAM, with secreted and SMG-specific genes up-regulated and Notch or progenitor genes down-regulated (Fig. 1f-g). Despite no detection of mature acinar and mucous cell markers in early long-term culture, adding DAPT for differentiation culture revealed more salivary gland-like features, suggesting the system maintained the cell heterogeneity, differentiation potential, and functions of mouse SMG organoids during long-term passage.

Fig. 1 Adult SMG organoids conserved various salivary glandular cell properties with secretory function in mice.

Then, mice PG and SLG organoids were generated using the same method as for SMG organoids. PG organoids had small tree-like ducts, SLG organoids had large cystic lumens (Fig. 2a). Immunohistochemistry results showed PAS-positive cells in SLG organoids' budding structures but not in PG organoids (Fig. 2b). AMY2 was mainly expressed in PG organoids, mucous cells and MUC19 in SLG organoids, consistent with salivary gland tissue expression patterns (Fig. 2c-d). TEM ultrastructural images showed serous or mucous granules in PG or SLG organoids' acinar cells (Fig. 2e). The overall gene expression profiles of the three major salivary gland organoids showed differential expression of related genes, indicating they retained tissue-specific characteristics at protein and transcript levels (Fig. 2f-h).

Fig. 2 Adult salivary gland organoids possessed distinct gland-specific properties in mice.

Based on the mice salivary organoid culture system, they adjusted the expansion medium with substances like prostaglandin E2 and established human PG, SMG, and SLG organoids that could grow and expand for 4 months (Fig. 3a-b). Adding DAPT and removing PGE2, nicotinamide, and CHIR99021 from GEM effectively differentiated the organoids (Fig. 3c). Intracellular calcium concentration in human SMG, PG, and SLG organoids rose, and swelling induced by neurotransmitters such as CCh, IPR, and VIP was detected (Fig. 3d-e). PAS and fluorescence staining showed mucin-positive lumens in SMG and SLG but not PG. MUC7 was in SMG and SLG, AMY1 only in PG and SMG, consistent with their source glands. These data suggest human salivary gland organoids have cellular heterogeneity, structural diversity, and tissue-specific functions.

Fig. 3 Establishment of human salivary gland organoids from PG, SMG, and SLG.

Human salivary gland organoids showed heterogeneity and lacked a distinct branched phenotype (Fig. 4a). SLG organoids were more cystic, while PG ones were compact solids (Fig. 4b). PGs had larger CD49f+CD26−; and CD49f+CD26+ was enriched in SLG (Fig. 4c-d). Basal-derived organoids were solid, and luminal-derived were cystic (Fig. 4e). Smaller luminal cells had a higher organoid-forming ability (Fig. 4e-g). These results imply that both luminal/basal progenitor cells exist in human salivary glands and the culture system can maintain their traits.

Fig. 4 Human luminal or basal progenitor-derived organoids maintained their growth and distinct characteristics.

Subsequently, single-cell RNA sequencing was done on the organoids and tissue samples. Results showed the organoids had multiple cell types in 8 clusters (Fig. 5a). Clusters 1 and 2 had high expression of basal (BNC1, KRT5) and progenitor (KRT15) markers. Cluster 2 also had high expression of cycling genes like CDK1, CCNB2, and CDCA2 (Fig. 5b). The DEGs in clusters 3 and 4 were highly expressed in acinar or luminal duct cells, with some unique genes (Fig. 5c, e), suggesting both clusters had acinar-like features. PG highly expressed LTF, and prolactin-induced protein or MUC7 were mainly in SMG or SLG respectively (Fig. 5f). In conclusion, some basal, luminal, acinar, or tissue-specific markers of salivary gland organoids were well maintained between tissues, and organoids.

Fig. 5 Single-cell RNA-seq revealed cellular heterogeneity and glandular diversity in human

adult salivary gland organoids.

To explore if the culture system could represent tumor behaviors, they observed organoid growth from benign or malignant tumors. Tumor organoids had different morphologies from normal ones, and their marker expressions were similar to tissues (Fig. 6a-e). Also, different tumor organoids showed sensitivity or resistance to nutlin-3 and various radiations (Fig. 6f-h). Thus, salivary gland tumor organoids have the potential to predict patient responses to drug or radiation therapies.

Fig. 6 Tumor-specific features were conserved in tumoroid models.

Conclusion

In conclusion, this study built long-term salivary gland organoid culture systems for mice and humans and optimized conditions. The organoids express specific genes and proteins and have glandular functions under neurotransmitter stimulation. Single-cell RNA sequencing shows human organoids' heterogeneity and diversity. Tumor organoids with specific traits are established, offering a platform for tissue regeneration and precision anti-cancer treatment research.