Fig 1: Establishment of human salivary gland organoids from PG, SMG, and SLG.a Schematic image of experiments using three major human salivary gland organoids, including human parotid gland (hPG), sublingual gland (hSLG), and submandibular gland (hSMG) organoids. b hSMG organoids were maintained in the GEM. The growth of a single organoid was tracked in the time-lapse of images obtained at each time point. Scale bars indicate 50 µm. c hSMG organoids were maintained in the GEM for 2 weeks and incubated in the DAM for another 3 days. Harvested organoids were subjected to H&E staining or IF staining for duct (KRT5; green, KRT7; red), acinar (AQP5; green, MIST1; red), and myoepithelial (ACTA2; green, KRT14; red) markers. Nuclei were stained with Hoechst 33342 (blue). Scale bars indicate 50 µm. d hSMG organoids were maintained for 3 months in the GEM, followed by differentiation in the DAM for another 3 days. Single-cell suspensions were prepared, and calcium influx was assessed using Fluo 4-AM in stimulation with either ATP (top) or carbachol (CCh; bottom) at different dosages. Red arrows indicate time points at which cells were treated with stimulants. e–g hPG, hSMG, and hSLG organoids were maintained for 1 month in the GEM, followed by differentiation in the DAM for another 3 days. e Differentiated hSMG organoids were stimulated with vehicle (DMSO), CCh, isoproterenol (IPR), or vasoactive intestinal peptide (VIP) for 1 h. Then, organoid swelling was observed under a brightfield microscope. Red arrows indicate swollen organoids. Scale bars indicate 500 µm. f Sections from hPG (top), hSMG (middle), and hSLG (bottom) tissues (left) and differentiated organoids (right) were subjected to PAS staining. Nuclei were counterstained with hematoxylin. Scale bars indicate 50 µm. g hPG, hSMG, and hSLG tissues (left) and differentiated organoids (right) were subjected to IF staining for mucous (MUC7, green) and serous (AMY1, red) acinar cells. Scale bars indicate 50 µm. All data were representative of three independent experiments. Source Data are provided as a Source data file.
Fig 2: Human luminal or basal progenitor-derived organoids maintained their growth and distinct characteristics.a Brightfield images of human PG, SMG, and SLG organoids cultured for at least 1 month in the GEM. Scale bars indicate 500 µm. b Organoids with solid or cystic morphology were counted and statistically analyzed (n = 4 biologically independent samples). c Single-cell suspensions were prepared from the three major human salivary gland tissues, and then assessed for the expression of CD49f and CD26 markers in epithelial cells via flow cytometry. d The percentage of CD49f+ CD26-, CD49f+ CD26+, and CD49f- CD26– populations were measured via flow cytometry and statistically analyzed (n = 4 biologically independent samples). The box and whisker plots indicate mean, SEM, and 5th and 95th percentiles. e, f CD49f+ CD26-, CD49f+ CD26+, and CD49f- CD26– cells from human SMG were isolated and cultured in the GEM for 3 weeks. Brightfield images were obtained for the assessment of organoid growth (e) and the evaluation of organoid formation efficiency (f). Scale bars indicate 200 µm (n = 5 biologically independent samples). g Organoids from CD49f+ CD26- and CD49f+ CD26+ cells were maintained in the GEM for 3 weeks and subjected to H&E staining or IF staining for CD49f (green)/CD26 (red), duct (KRT5; green, KRT7; red), acinar (AQP5; green, MIST1; red), and myoepithelial (ACTA2; green, KRT14; red) markers. Nuclei were stained with Hoechst 33342 (blue). Scale bars indicate 50 µm. Data are representative of at least three independent experiments and presented as mean ± SEM. *p < 0.05, ***p < 0.001, n.d, not detected. Source data are provided as a source data file.
Fig 3: Mist1-K8.18-PCNA, K5-PCNA co-labelling: in general, the control SMGs showed a low rate of cellular turn-over. An overall increase in the number of PCNA-positive cells was seen in the early regeneration phases following poly (I:C) injection (72 and 96 hrs P-PIC) (acinar cells: orange arrows, duct cells: blue arrows). While abundant PCNA co-localization at 72 hrs was seen with K5 progenitor cells (arrows), at 96 hrs the proliferating cells were mostly acini (n > 20 fields from three independent animals: 20x in the Mist1-PCNA-K8.18 experiments and 40x in the K5-PCNA experiments). Scale bar = 10 µm.
Fig 4: Acute injury/regeneration SG model: (a) Illustration depicting poly (I:C) retrograde ductal injection: 20 µl of poly (I:C) (4 µg/µl) was injected via an insulin syringe into the C57BL/6 SMG via Wharton’s duct. (b) Measurement of pilocarpine-stimulated saliva: retro-ductal injection of the vehicle (V-C) did not affect SMG saliva flow rate. Conversely, post poly (I:C) (P-PIC), saliva secretion was promptly lost, but was rapidly restored after 72 hrs following the innate immune stimulant. Note the progressive functional recovery after 96 hrs, 7 and 14 days (n = 5–14 gland/group). (c) SMG H&E analysis: the control SMGs (V-C) revealed normal compact parenchyma formed of secretory acini and collecting ducts. At 24 hrs, in addition to the inter-and intra-lobular infiltration of immune cells triggered by poly (I:C), acini were enlarged and occasionally devoid of nuclei (arrows). After 72 hrs (initial recovery of salivation), ducts and duct-like structures prevailed in the SMGs, with obvious reduction in the acinar phenotype. The histomorphology dramatically changed at 96 hrs following poly (I:C) injection: newly emerging, basophilic acinar cells were globally seen in the regenerating SMGs. Scale bar = 20 µm. (d) Transient loss and rapid regain of the acinar phenotype was confirmed using the acinar differentiation marker Mist1. Approximately, 50% acinar reduction was observed, 24 hrs post innate immune challenge. This prompt depletion became more extensive (80%) at 72 hrs. Surprisingly, after 96 hrs of poly (I:C) challenge, the SMGs were significantly re-populated by Mist1-positive cells, the percentage of which approached the control values (mean ± SD, n = 15 fields (20×)/group, ****P < 0.0001 and *P = 0.05). Scale bar = 20 µm. (e) IHC and PCR of AQP5 and NKCC1: extreme loss of these key functional determinants 24 hrs P-PIC and their incomplete recovery at the 96 hrs time point which featured enhanced, control-like expression of acinar Mist1. Scale bar = 20 µm.
Fig 5: Adult submandibular gland (SMG) organoids conserved various salivary glandular cell properties with secretory function in mice.a Schematic image of experiments using the three major murine salivary gland organoids, including murine parotid gland (mPG), murine sublingual gland (mSLG), and murine submandibular gland (mSMG) organoids. b mSMG organoids were maintained in the growth expansion medium (GEM) or GEM lacking each factor, and their growth was observed via brightfield microscopy. Box indicates a representative organoid. Scale bars indicate 500 µm. c A mSMG organoid at early passage (p1) was maintained in the GEM and tracked in time-lapse of the images obtained at each time point. Scale bars indicate 100 µm. d, e mSMG organoids were maintained in the GEM for 9 days or further incubated in a differentiation-accelerating medium (DAM) for another 3 days. d mSMG organoids were subjected to hematoxylin & eosin staining (H&E, left), immunofluorescence staining (IF) for ductal markers (middle, KRT5; green, KRT7; red), or periodic acid-Schiff staining (PAS, right). Red arrows indicate PAS-positive regions. Nuclei were stained with hematoxylin (H&E and PAS) or Hoechst 33342 (IF). Scale bars indicate 100 µm. e The expression of epithelial (left, TJP1; green, CDH1; red, CC3, cleaved caspase-3; white), acinar (middle, AQP5; green, MIST1; red), and myoepithelial (right, ACTA2; green, KRT14; red) markers was assessed. Nuclei were stained with Hoechst 33342 (blue). Scale bars indicate 50 µm. f The expression of salivary gland markers and proliferation-related genes in mSMG organoids cultured under GEM or DAM conditions with SMG tissues (n = 2). Heatmap illustrates TMM-normalized expression values. g Heatmap of differentially expressed genes (fold change >2, p < 0.05) between murine organoids grown under GEM and DAM conditions with SMG tissues (n = 2). Heatmap illustrates row z-score of TMM-normalized expression values. All data were representative of three independent experiments.
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