Limiting Dilution Technique for Salivary Gland Stem Cell Isolation

Objective:Annually, 500,000 new cases of head and neck cancer occur worldwide. Most of these patients are subjected to irradiation (IR) resulting in irreversible damage to the salivary glands (SG). At present, biological oriented treatments for these patients are absent. Recent studies support the hypothesis that adult SG contains epithelial stem-cells such as integrin-á6â1-expressing-cells (SGIE). Such autologous cells can be isolated, cultivated, and used for implantation into the previously irradiated gland to regenerate SG function. A main concern with this model is re-implanting a heterogeneous population of cells that may allow infiltrating of tumorigenic cells, derived from the host or due to abnormal growth in culture back to the patient. Hence, our objective is to develop a method that provides homogeneous SGIE culture, allowing reliable tumorigenic-transformation-tests and safe auto-transplantation. Methods: Rat SGIE from submandibular SG were secluded by Percoll gradient-technique followed by MACS methodology, and cultured for several passages on collagen type-1. To isolate colonies derived from a single cell, a limiting dilution assay was utilized: cells were diluted in the medium to 0.5cells/well and seeded on 96-well plate. To examine in-vivo characterization, cells were implanted into rat SG subjected to irradiation Results: Clones were successfully derived from a single SGIE. Clones preserved a normal 2N karyotype and could not produce colonies on soft-agar test, implying non-tumorigenic phenotype. Furthermore, these cells maintained progenitor differentiation abilities. When clones implanted into H&N irradiated rats to the SG, these cells restored saliva secretion to almost a normal level. Conclusions: These findings are encouraging toward accomplish our goal to establish a stem-cells-based auto-transplantation treatment to regenerate irradiated SG's dysfunction.