Salivary gland organoid transplantation as a therapeutic option for radiation-induced xerostomia

Abstract

Background: Xerostomia is a pathological condition characterized by decreased salivation due to salivary gland dysfunction and is frequently attributed to irreversible damage as a side effect of radiation therapy. Stem cell-derived organoid therapy has garnered attention as a promising avenue for resolving this issue. However, Matrigel, a hydrogel commonly used in organoid culture, is considered inappropriate for clinical use due to its undefined composition and immunogenicity. In this study, we aimed to develop a method for culturing collagen-based human salivary gland organoids (hSGOs) suitable for clinical applications and evaluated their therapeutic effectiveness.

Methods: Human salivary gland stem cells were isolated from the salivary gland tissues and cultured in both Matrigel and collagen. We compared the gene and protein expression patterns of salivary gland-specific markers and measured amylase activity in the two types of hSGOs. To evaluate the therapeutic effects, we performed xenogeneic and allogeneic transplantation using human and mouse salivary gland organoids (hSGOs and mSGOs), respectively, in a mouse model of radiation-induced xerostomia.

Results: hSGOs cultured in Matrigel exhibited self-renewal capacity and differentiated into acinar and ductal cell lineages. In collagen, they maintained a comparable self-renewal ability and more closely replicated the characteristics of salivary gland tissue following differentiation. Upon xenotransplantation of collagen-based hSGOs, we observed engraftment, which was verified by detecting human-specific nucleoli and E-cadherin expression. The expression of mucins, especially MUC5B, within the transplanted hSGOs suggested a potential improvement in the salivary composition. Moreover, the allograft procedure using mSGOs led to increased salivation, validating the efficacy of our approach.

Conclusions: This study showed that collagen-based hSGOs can be used appropriately in clinical settings and demonstrated the effectiveness of an allograft procedure. Our research has laid the groundwork for the future application of collagen-based hSGOs in allogeneic clinical trials.

Keywords: Organoid; Organoid transplantation; Salivary gland; Stem cell therapy; Xerostomia.

Fig. 1

Generation of hSGOs in Matrigel-based culture and scRNA-seq analysis. (A) Microscopic image of the growth of hSGOs cultured in optimized medium and stained with Hematoxylin and Eosin (H&E) on day 5 of passage 5 (Left). Scale bar, 100 μm. Measurement of size of organoids (μm²) on day 5 and 10 (Right). n = 4, biologically independent samples. **** p < 0.0001 (B) Cumulative cell number of passage 0 to 9. n = 3, biologically independent samples. (C) Immunofluorescence staining of proliferative cell marker Ki67 in hSGOs on day 5 of passages 2, 6, and 12 (Left), percentage of Ki67-positive cells in hSGOs (Right). Scale bar, 100 μm. (D) Uniform manifold approximation and projection (UMAP) analysis of human submandibular gland tissue (n = 1,965 cells). (E) The expression of acinar cell markers (AQP5, LPO, AMY1A, and AMY2B) within the acinar cell clusters. (F) The expression of ductal cell markers (KRT19, WFDC2, KRT5, and KLK1) within the ductal cell clusters. (G) UMAP analysis of hSGOs at day 5 of passage 5 (n = 5,220 cells). (H) The expression of ductal cell markers (KRT19, WFDC2, and KRT5) within the ductal-like cell clusters, (I) Acinar cell marker (AQP5) within the acinar-like cell cluster, and (J) Cycling cell markers (CDK1 and MKI67) within the cycling cell clusters

Fig. 2

Differentiation and functional replication of hSGOs cultured in Matrigel. (A) Scheme of differentiation of hSGOs. On day 5, the expansion medium (EM) was replaced with differentiation media (DM), and the organoids were cultivated for an additional 10 days. (B) Microscopic images of organoids cultured in EM and DM (Left). Size of hSGOs in the EM and DM (Right). Scale bar, 100 μm. n = 4, biologically independent samples. *** p < 0.001. (C) Gene expression of acinar cell, ductal cell, and functional relevance markers of hSGOs on days 5 in EM (Undifferentiated) and day 15 in DM (Differentiated) (Left). Relative gene expression levels of AQP5, AMY1A, and CRISP3 (Right). n = 5 or 6, biologically independent samples. * p < 0.05, ** p < 0.01, **** p < 0.0001. (D) Immunofluorescence staining of acinar cell markers (AQP5 and AMY) and ductal cell markers (KRT19 and KRT7) in hSGOs on day 15 in EM and DM. Scale bar, 100 μm. (E) H&E and periodic acid-Schiff (PAS) staining of hSGOs on day 15 in EM and DM. Scale bar, 100 μm. (F) Amylase activity of hSGOs cultured on days 5 (Undifferentiated) and days 15 (EM and DM). n = 4, biologically independent samples. ** p < 0.01. (G) Calcium influx measurement using Fluo-4 AM in hSGOs on day 15 in DM following stimulation with 50 mM carbachol. Scale bar, 100 μm

Fig. 3

Generation and characterization of collagen-based hSGOs. (A) Microscopic image of the growth of hSGOs cultured in a type I collagen matrix. Scale bar, 100 μm. (B) Organoid diameters at days 4 and 7 of passages 1 to 4. (C) Cumulative cell number of passage 1 to 8. n = 3, biologically independent samples. (D) Gene expression of the acinar cell markers (AMY1A, AQP5, and CRISP3) and ductal cell markers (KRT19, KRT7, and WFDC2) of undifferentiated and differentiated organoids. n = 3 or 4, biologically independent samples. * p < 0.05. (E) Immunofluorescence staining for acinar cell markers (AQP5, AMY, CD166, and MUC5B) and ductal cell markers (KRT7 and KRT19) in undifferentiated and differentiated hSGOs and tissue. Scale bar, 100 μm. (F) H&E and PAS staining in undifferentiated and differentiated hSGOs and tissue. Scale bar, 100 μm. (G) Measurement of amylase activity of undifferentiated and differentiated hSGOs. n = 4, biologically independent samples. *** p < 0.001

Fig. 4

Transplantation of collagen-based hSGOs in a xerostomia mouse model. (A) Strategy for transplantation of hSGOs to a radiation-induced xerostomia mouse model. Scale bar, 250 μm. (B) Engrafted region of parotid gland organoids labeled with Cytopainter at 2 and 4 weeks (Left, white arrowhead indicate an engrafted region). Historical analysis revealed large nuclei-consisting organoids in the recipient’s submandibular gland at 4 weeks post-transplantation (Right, yellow dashed line). Scale bar, 100 μm. *** p < 0.001. (C) Human E-cadherin, human nucleoli, KRT19, CD166 were detected in the engrafted organoid of the recipient’s submandibular gland by immunofluorescence staining. Scale bar, 100 μm. (D) Immunofluorescence staining with MUC5B and Alcian blue staining for mucin secretion. Scale bar, 100 μm. (E) Historical analysis of integrated organoids on 16 weeks post-transplantation (Yellow dashed line). Scale bar, 100 μm. (F) Expression of human E-cadherin, KRT19, and (G) MUC5B in engrafted hSGOs at 16 weeks post-transplantation. Scale bar, 100 μm. (H) Alcian blue staining for mucin secretion in engrafted hSGOs at 16 weeks post-transplantation. Scale bar, 100 μm

Fig. 5

Generation and structure recapitulation of mSGOs. (A) Microscopic image of the growth of mSGOs on days 6, 9, and 12 of passage 0, 6, and 9. Scare bar, 100 μm. (B) H&E staining of mSGOs on days 6 and 12 of passage 0 and 6 for identifying the acini and duct structure. Scale bar, 100 μm. (C) PAS staining of mSGOs on days 6 and 12 of passage 6 for identifying the secretion of mucin (Red arrowhead). Scale bar, 100 μm. (D) Immunofluorescence staining for acinar cell marker (Aqp5), ductal cell markers (Krt17 and Krt14), and proliferative cell marker (Ki67) in mSGOs on days 6 and 12 of passage 0 and 6. Scare bar, 100 μm. (E) Calcium signaling assay of mSGOs on day 12. Intracellular calcium mobilization was observed after stimulation with 50 mM carbachol. Scale bar, 100 μm

Fig. 6

Transplantation of mSGOs in a xerostomia mouse model. (A) Strategy for transplantation of EGFP-mSGOs cells in radiation-induced xerostomia model. (B) Visualization using fluorescein paper for saliva secretion at 8 weeks post-radiation. Fluorescence intensity was measured by opening the mouth using tweezers (Left). Graph shows the measurement of saliva secretion by time (Right). n ≥ 3 mice per group. Mean ± SD. *, Control vs. IR, p < 0.05; #, IR vs. IR-TP 10⁵, p < 0.05. IR, irradiation; IR-TP 10⁴ and 10⁵, irradiation-transplantation and doses (cell number). (C) Representative gross image of salivary glands at the age-matched controls and at 16 weeks post-radiation (Left). Comparison of salivary gland tissue weight (Right). n ≥ 3 mice per group. Bar, mean ± SD. **, Control vs. IR, p < 0.01; ***, IR vs. IR-TP 10⁴, p < 0.001. (D) A comparison of histological analysis using H&E staining. Salivary gland tissues from the age-matched controls and at 16 weeks post-radiation. Scale bar, 50 μm