Salivary gland (SG) dysfunction results in limited or complete loss of saliva production, leading to xerostomia. This is caused by both complications of autoimmunity (Sjogren’s Syndrome) and side effects from cancer radiation therapy. The cells most affected are the acinar cells, which are responsible for producing and secreting saliva, and with chronic illness caused by radiation therapy, these cells cannot recover by themselves. Without saliva, patients not only experience loss of lubrication in the oral cavity, but also decreased immune defense and tissue loss due to the reduction of salivary antimicrobial properties, leading to a decreased quality of life and risk for systemic infection. Current treatments are superficial, including artificial saliva, which provides only temporary relief and fail to address the core of the issue; however, regenerative medicine may enable more efficient and permanent solutions toward regaining SG function. To this end, we have developed a novel differentiation strategy based on salivary gland (SG) development to obtain SG epithelial progenitors from induced pluripotent cells with no need for virus-mediated gene overexpression or embryoid body microsurgery. In this study, we aimed to determine how these SGEPs can result in polarized organoids which can be matured and made functional in vitro, as well as contribute to salivary gland tissue regeneration in vivo.
Methods:
iPSCs were cultured until 70% confluent before differentiation commenced. The cells were subjected to a sequence of differentiation steps mimicking SG development. On day 17 (D17) cells were seeded into Cultrex which was allowed to gel at 37oC and maintained in culture for another 30 days in differentiation media that were designed to enable stepwise differentiation. Organoids maturation was assessed with a battery of methods including live cell imaging using a Sox10-GFP reporter, RT-PCR, immunostaining and RNA sequencing. Development of salivary function was determined with a surrogate assay measuring calcium ion flux in response to carbachol stimulation. In addition, vivo transplantation experiments were designed to determine the ability of these cells to form SG tissue in vivo; as well as the stage of differentiation that yields optimal SG regeneration. To this end, D17 progenitor cells and D32 organoids were implanted into the athymic mouse submandibular gland (SMG) that was subjected to excisional wounding. After 6 and 12 weeks, mice were sacrificed and SMGs were collected. The tissues were fixed in 4% paraformaldehyde and embedded in paraffin before being sectioned and prepared for histological analysis to determine to what extent the cells and organoids contributed to salivary gland regeneration.
Results and Conclusion:
After 17 days of differentiation, cells expressed key markers of SGEP progenitors, such as FoxD3 and Sox9, as evidenced by RT-PCR and immunostaining. We employed the SGEPs to develop luminal SG organoids (SGO) in 3-dimensional hydrogels. We observed lumenization after one week in culture, and upregulation of acinar progenitor markers after two weeks, indicating acinar differentiation. After 30 days in 3D culture, we observed the presence of polarized SGO, expressing mature acinar markers. Furthermore, we developed a novel strategy to obtain SGO with salivary function, as evidenced by response to the acetylcholine analogue, carbachol. We also discovered that SGO maturation depended on the mTOR2 signaling pathway, using a combination of chemical inhibition and genetic knockdown strategies. Notably, transplantation of mCherry+ human SGEP progenitors or SGO into a mouse model of wounded SG showed that SGEPs yielded a hypertrophic and disorganized SG. On the other hand, transplanted SGOs gave rise to organized SG containing acinar, ductal, and myoepithelial cells, reminiscent of native and functional SG, as evidenced by histology and immunostaining for lineage specific markers.
In conclusion, our work is the first to derive SGEP and SGO from hiPSC without gene overexpression or organoid microdissection and may have significant impact in understanding SGO growth and maturation. In vitro applications include the potential use of SGO/salispheres for drug screening and testing as well as disease modeling. In vivo applications include the development of cell therapies to treat hyposalivation disorders and other causes of salivary gland dysfunction.