Toward Salivary Gland Stem Cell Regeneration

Head and neck cancer is the sixth most common cancer worldwide, resulting in ~ 640,000 cases and ~ 350,000 deaths per year. Most of these patients receive irradiation treatment.¹ This treatment modality has a pronounced effect on the salivary glands, resulting in irreversible damage to the parenchymal tissue.² Patients who have received irradiation treatment have rampant dental caries, frequent mucosal infections, and difficulties swallowing and chewing dry food. These patients are also sensitive to spicy food, have alterations in taste sensation and perception, and experience considerable pain originating in the impaired salivary glands, coughing episodes, voice disturbances, speech difficulties, and discomfort, all of which significantly decrease quality of life.³

Therapeutic Model

In recent years, the authors of this article and other researchers have concentrated on methods to restore normal salivary glands function in these patients.^4,5 The notion of salivary gland regeneration is based on the assumption that autologous salivary gland graft cells can be isolated before initializing irradiation, cultivated, and preserved during the irradiation period. After treatment, cells are implanted into the irradiated gland and replace the functionally damaged cells (Figure 1).

Salivary Gland Stem Cells

The specific mechanisms involved in the physiological regeneration of salivary cells are still unknown, although regeneration from a putative noncommitted stem cell population is preferred.⁶ To date, the specific tissue stem cells remain unidentified and their exact location within the salivary glands remains obscure.⁷ Many nuclear, cytoplasmic, and cell surface markers were suggested to characterize salivary gland stem and progenitor cells (Table 1). Numerous studies hypothesize that salivary gland progenitor cells are located among the intercalated duct basal cells, as they have been shown to have a high proliferating capacity.^6,8-11 These basal cells can migrate horizontally or luminally during differentiation (Figure 2). Other groups supporting this notion showed that tissue damage induced by ligation of the main salivary gland ducts leads to severe atrophy of the acini and granular ducts. The reopening of the ligated ducts results in marked proliferation of the ductal cells, which leads to repopulation of the acinar cell region.^12,13 Integrin α6β1, which is part of the hemidesmosome complex and is responsible for the adhesion to extracellular laminin, has been suggested as a specific basal cell surface marker for isolation of salivary gland progenitor cells.^13,14 The integrin α6β1–positive cell population was reported as possible progenitor cells mainly for liver and pancreatic lineages¹³ after isolating them 6 days for duct ligation, salivary gland removal, and cultivation. c-Kit (CD117) is a tyrosine kinase receptor, the expression of which is a common trait of stem and precursor cells in the organism, suggesting that it may also be a key in conferring and maintaining stemness and high self-renewal potency.^15-17 The α6β1-positive cell population was shown to be positive for cell-surface markers such as Sca-1 and c-Kit as well.¹⁸

Several methods, including selection utilizing a growth matrix,^13,19 specific growth conditions,²⁰ and salispheres,^1,5 have been described for isolation and culture of the salivary gland stem cells. As mentioned, salivary tissue consists of different cells, such as parenchymal cells, fibroblasts, and blood cells. Therefore, primary cultures of dispersed cells will always contain the entire repertoire of cell types, making it difficult to choose a specific salivary gland cell⁷ and will be restricted to a relatively limited culture lifespan during in vitro cultivation, resulting in a narrow time window for implantation. Previously, the authors of this article described a methodology for the isolation of salivary cells expressing integrin α6β1 from adult rodents using magnetic affinity cell sorting technology, which resulted in a very high-purity cell fraction of a putative progenitor cell population termed salivary gland integrin α6β1 positive cells (SGIE).^21,22 Other research groups, such as Coppes et al, use different cell surface markers such as c-Kit, Sca-1, prominin-1, and integrin α6β1 to enrich the epithelial progenitor cells in the salivary glands.¹⁶

Irradiation Mechanism

Radiosensitivity of salivary gland acinar cells was considered for a long time an enigmatic behavior as these cells are nondividing and well differentiated with a structure and function similar to exocrine pancreas cells.² Unlike other radiosensitive tissues, salivary gland cells proliferate slowly and are highly differentiated. Thus, the effect of irradiation cannot be attributed solely to rates of tissue proliferation. In 1984, Abok et al²³ suggested that irradiation of salivary glands causes membrane damage to the secretion granules in the secreting cells. This results in proteolytic enzymes leakage, causing the cells to disappear. However, Konings et al² reported in 2005 that after 15 Gy irradiation, no change was in the population of acinar cells both in parotid and submandibular glands a few days after irradiation, indicating that cell disappearance due to granular leakage is not substantial.² One of the main theories suggests that the irradiation results in sublethal DNA damage, which manifests and becomes lethal at a delayed phase. Thus, when salivary progenitor cells are reproducing and parenchymal replenishment is needed, they die.²⁴ DNA damage to progenitor cells and stem cells causes insufficient cell renewal.^2,25

The authors examined the effects of irradiation on salivary glands²⁶ in order to investigate and define the molecular mechanisms affecting salivary glands following irradiation, directing efforts on saliva proteome and global transcription profile of submandibular salivary gland (SSG) tissue. Saliva protein patterns were studied by proteomic analysis following mass spectrometry analysis that revealed proteins originating from SSG that were reduced in expression and proteins derived from the serum that exhibited increased expression, both indicating salivary tissue damage.²⁶ The increase in serum proteins suggests irradiation damaged the extracellular environment, causing loss of barrier function between serum and salivary glands, whereas the decrease in salivary gland–derived proteins suggests damage to the protein synthesis, processing, or trafficking pathways. To examine whether irradiation causes alterations in mRNA expression, microarray analysis was performed, followed by validations. The authors found significant alterations in cell cycle–regulating genes in which 4 were found to be affected by irradiation: cyclin D1, p21, p57, and Ywhaq.²⁶ Following irradiation, most of these genes showed a major up-regulation, which suggests that a cell cycle-arrest response occurs after irradiation.²⁶ c-Kit–expressing cells have been suggested to belong to the salivary gland cell-dividing fraction.¹⁵ To further validate cell proliferation and cell arrest, the authors examined c-Kit transcription expression in vivo and the number of SGIE cell colonies²¹ obtained from the SSGs before and after irradiation. The authors found that they both were significantly decreased, indicating damage to the progenitor cells and stem cells, causing insufficient cell renewal and cells’ proliferation abilities.^2,24,26

Salivary Gland Regeneration

At present, the major restriction in handling salivary gland stem cells, as also reported for c-Kit expressing salivary stem cells,¹⁵ is their relatively limited lifespan during in vitro cultivation, resulting in a narrow time window for implantation. To overcome this difficulty, the authors established a personal autologous salvary gland stem cell bank (Figure 1).²⁷ Personal stem cell banking may be important for the repair process by autologous transplantation due to the long time frame (approximately 6 weeks or more) of the irradiation treatment that might result in reduced implantation efficiency. SGIE cells were cryopreserved for up to 3 years and evaluated for genetic and functional stability. No alterations were found, indicating no tumorigenic changes due to the cryopreservation–thawing–reculturing process.²⁷

Another important issue is the safety of the implantation in regard to future clinical applications. Using cultures derived from a single cell rather than from cells originated from a mixture of cell clones enables better evaluation for whether a representative cell sample is expressing a tumor-related phenotype. For that reason, the authors created a novel methodology for the production of single cell–based salivary stem cell clones,²⁷ which is essential for safety assurance of future clinical implantation.

Salivary gland stem cells were isolated from salisphere cultures and were transplanted into murine-irradiated SSGs.^15,28 This transplantation resulted in 70% recovery of salivary flow rate, when implanted with 300 c-Kit donor cells.¹⁵ When non–c-Kit cells were implanted, only 33% recovery of salivary flow rate was reported. The regenerative capacity of other salivary gland stem cells-expressing markers such as α6β1, and CD133 was examined. The result was functional recovery of the saliva flow. Although these animal salivary models show promising results, confirmatory data in humans are required and further studies and technologic fine-tuning are needed before clinical implementation.

About the Authors

Raluca Stiubea-Cohen, MSc, PhD cand.
PhD student
Faculty of Dental Medicine
Institute of Dental Sciences
The Hebrew University of Jerusalem
Jerusalem, Israel

Ran David, MD, PhD
Faculty of Dental Medicine
Institute of Dental Sciences
The Hebrew University of Jerusalem
Jerusalem, Israel

Yoav Neumann, MSc, PhD cand.
PhD student
Faculty of Dental Medicine
Institute of Dental Sciences
The Hebrew University of Jerusalem
Jerusalem, Israel

Aaron Palmon, DMD, PhD
Professor
Faculty of Dental Medicine
Head, Institute of Dental Sciences
The Hebrew University of Jerusalem
Jerusalem, Israel

Doron J. Aframian, DMD, PhD
Associated Professor
Head, Salivary Gland Clinic and Saliva Diagnostic Laboratory
Department of Oral Medicine
The Hebrew University of Jerusalem
Jerusalem, Israel
Hadassah Medical Center
Jerusalem, Israel

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*These two authors contributed equally to this work.