Salivary Gland Stem Cells and Tissue Regeneration: An Update on Possible Therapeutic Application

INTRODUCTION

SCs therapy has become a wide field for research since the discovery of their pluripotency. Concerning SG regeneration, SCs could be a promising treatment for xerostomia resulting from SG hypofunction. SG damage due to radiation, autoimmune diseases like Sjorgen’s syndrome¹ or metabolic syndromes like diabetes,² and age-related degeneration are the common etiology for SG impairment. The reduction of saliva has severe effects on the patient’s quality-of-life as this deteriorates taste perception, swallowing, and speech performance. It also leads to the increase in the incidence of dental caries, mucosal atrophy, and ulceration, in addition to an overall reduction of immune response against infectious agents. This may be critical in case of immunocompromized patients as dissemination of fungal, viral, or bacterial infections can be life-threatening.¹ Since there is still no efficient treatment, SG regeneration is anticipated and an SC-guided transplantation and differentiation with the assistance of scaffolds and growth factors within affecting SG could be an ideal option.³ Apart from their own regeneration, SG, especially the minor glands found in the oral mucosa, could be an effective alternative and an easily accessible source of SG SC (Fig. 1) for the regeneration of other organs like the liver⁴ and the pancreas⁵ based on the morphological, embryological, and functional similarities.

The special characteristic of SCs is their ability to differentiate into any other cell type through mitosis and, for unlimited times, as long as they are provided with the right stimulus.⁶ Adult SCs operate as an internal repair system and are found in various tissues.⁷ Over the last decade, scientists studied the induced pluripotent SCs (iPSCs) that are adult cells that could be “reprogrammed” genetically to assume an SC-like state.⁸ SC populations bearing embryonic or adult SC characteristics have been identified in human major SGs (parotid and submandibular), within their ductal system.^9,10 In humans, minor SGs the single epithelial SCs as well as SCs with both epithelial and mesenchymal characteristics have been isolated only recently.^11,12

HISTOLOGY AND EMBRYOLOGY OF SGS

There are major and minor SGs in the head and the neck area.¹³ The major SGs include the parotid (which is the largest), submandibular, and sublingual glands. SGs consist of the cells epithelial in nature, secretory called acinar, myoepithelial cells, and ductal system. The saliva is produced at the level of acini and passes through the ductal system which is composed of intercalated, striated, and excretory ducts. The intercalated ducts and the basal cells of the striated duct have been speculated as a possible position of SCs.

A conflict concerning the embryologic origin of the epithelium of SGs divides researchers. Specifically, citations refer that the parotid originates from the ectoderm, whereas the submandibular and sublingual glands derive from the endoderm. Concerning minor SGs, their origin is basically unknown, except for Von Ebner minor SGs which are originated from the endoderm. However, Rothova et al. used mice and Sox17-2A-iCre/R26R reporter for tracing endoderm progenitor cells during oral development.¹⁴ In contrast to previous studies, Rothova et al. suggested that major SGs originate from the ectoderm. Palatal minor mucous glands have both endodermal and the ectodermal origin, while minor lingual SGs may be derived from the endoderm.¹⁴ However, it is important to perform lineage tracing with endodermal Cre drivers to confirm speculations.¹⁴ Labial SGs (LSGs) are mixed, mostly mucous, and are situated mainly in the center of the oral cavity.¹²

REVEALING THE EXACT LOCATION OF SCS

The presence of SCs within SGs is a controversial subject for more than a decade. SCs in the major SGs have been identified through labeling of cells with bromodeoxyuridine (BrdU). The population of label-retaining cells (LRCs), which did not express keratin 18 (an acinar/duct cell marker) or α-smooth muscle actin (myoepithelial cell marker), were considered to be the SC population. Those LRCs were specifically explored at the top of the intercalated duct but some were also observed in the excretory duct.¹⁵

In 2014, Andreadis et al.¹² isolated a mixed cell population from normal human labial minor salivary glands (MSGs). The population were characterized with SC properties of the mesenchymal and epithelial features¹² (Fig. 2). The superficial location of minor SGs in the lip could consist of an innovative source of adult SCs without any ethical limitation.¹² A group of scientists confirmed the presence of human MSG SCs (hMSG MSCs) in the lamina propria of oral mucosa.¹⁶ Recently, these have been observed on the basal layer of the excretory duct and in the mesenchymal tissue between acinar and ductal epithelial structures of MSGs. They are characterized by enhanced SC capabilities with the potential to participate in tissue engineering.¹⁶

USE OF SC MARKERS

SC markers are genes and their protein products that can be used to characterize various SC populations. The field of SG SC research deciphered a plethora of putative markers that have been used to identify stem/progenitor cells, but there is evidence that only a few marker positive cells have the ability to actively restore irradiated glands.¹⁷ SC markers are also the key point to determine, which SC population could provide maximum regeneration, to achieve the best results with minimum tissue demands.

CKIT-CD117 (cell membrane protein tyrosine kinase receptor) has been detected in the excretory duct of human major SGs.¹² On the one hand, various studies have proved that c-kit+ cells are capable of proliferation and differentiation in vitro and in vivo.¹⁸ According to Lombaert et al., transplantation of only 300 c-kit+ cells was enough to rescue SG dysfunction in a mice model.¹⁸ This in vivo transplantation study points that c-kit+ cells show more SC-like characteristics than cells positive for other markers (CD133, CD49F, and CD24).¹⁸ On the other hand, mSG glands were negative for the CKIT-CD117 marker and, in addition, CKIT-CD117+ cells in cell lineage from adult mice SGs are heterogeneous and differentiated. Long-term in vivo lineage tracing showed at least two CKIT-CD117 ductal cell lineages, which did not play a crucial role in cell maintenance. These results question the reliability of CKIT-CD117 as a true SG SC marker.¹⁷

Nanog and Oct3/4 are embryonic SC markers, detected in major SGs.¹⁹ Interestingly, a similar pattern of Oct4 expression is also noticed in some kidney tubular cells, suggesting that they could be possible renal SCs, indicating possible similarities with SGSCs.¹⁹

Aldehyde dehydrogenases (ALDHs) are highly expressed in SCs and they participate in many biological activities. Various murine and human SCs like hematopoietic, neural, muscle, hepatic, and adipose SCs are found with elevated ALDH1 levels. Interestingly, human and mouse MSG SCs are characterized by a higher expression of ALDH3 rather than ALDH1. Higher levels of ALDH3 were found in SCs in comparison with those in non-SCs.²⁰ Another lineage tracing study has shown that ductal cells express Ascl-3, a transcriptional factor localized primarily to the ductal cell, and also express Nanog and Oct 3/4.²¹

Nestin is an intracellular SC marker expressed in cells undergoing proliferation and migration. It is strongly detected in human minor salivary gland mesenchymal stem cells (hMSGMSCs), implying its value in tissue engineering. Interestingly, in the human pancreas and SG SCs population, a similar pattern of nestin expression is seen.¹⁷ Integrin a6b1 is a proven SG progenitor cell marker in rats. These cells’ profile was expressed after duct ligation, and a6b1+ cells were able to generate both acinar-like and duct-like structures. However, these cell lines were characterized by uncontrolled growth, so further research is required before they could be used in vivo.⁸

SOX2 is a transcription factor that has been considered to regulate pluripotency in various epithelial tissues. During fetal development, SOX2 plays an important role in the formation of cell types, especially in the endoderm and the nervous system. In adult sublingual glands, SOX2+ cells are the putative SCs²² (Table 1). Interestingly, markers of SG SCs are also expressed in various SG malignancies like mucoepidermoid carcinoma.

AVAILABLE SCAFFOLDS FOR TISSUE ENGINEERING

Scaffolds are a really important in tissue engineering as they function as an extracellular matrix where cells adhere, proliferate, and differentiate. Therefore, scaffolds must be biocompatible and biodegradable (if necessary), so that they resemble the microenvironment of native tissue. In the presence of a scaffold, extracellular proteins and growth factors would regenerate functional organs. There has been much research on scaffolds that would be suitable for SG regeneration. Scaffolds can be subdivided into biologic (collagen, fibrin, silk, chitosan, alginate, and hyaluronic acid (HA)) and biocompatible (polyglycolic acid, poly-lactic acid, poly-lactic-co-glycolic acid, and polyethylene glycol) scaffolds. In addition, the biomaterial can be applied to 2D cultures and 3D cultures. In the first category, cells are cultured as a monolayer lining a tube consisting of polymers, whereas 3D cultures resemble acinar and ductal structures as cells are cultured in hydrogel matrices. For example, chitosan, a substance found in the exoskeleton of crustaceans, resembles glucosamine and has been proven to be capable of ensuing branching morphogenesis.²³ Furthermore, chitosan was chemically coupled to nano-fiber scaffolds and it was demonstrated that they could lead to increased proliferation compared to nano-fibers themselves and, at the same time, without cytotoxicity.²⁴ Matrigel is another potential scaffold which is derived from Engelbreth–Holm–Swarm (EHS) mouse sarcoma cells, which contain not only basement membrane proteins such as collagen IV, laminin, fibronectin, entactin, and perlecan but also multiple angiogenic and growth factors that regulate cell growth and differentiation.²⁵ This product has achieved successful results but only in vitro and is speculated as unsuitable for clinical translation as its components are not xeno-free and the basement membrane proteins derived from mouse sarcoma do not meet the requirements of the current good manufacturing practice (GMP) regulations by the FDA.²⁶

Noteworthy, a whole de-cellularized organ has been considered as an ideal bio-scaffold. The de-cellularized scaffold contains only its extracellular matrix proteins and native structures, but without any cellular components. It could adhere to the cells and lead to the expression of some differentiation markers, highlighting it as another potential scaffold for SG regeneration.²⁷

Polymers can also be used as substrates for SG regeneration, but scaffolds without extracellular matrix coating cannot provide cell adherence or growth. Shin et al. conducted a study in which human parotid epithelial cells were cultured on matrigel, on poly ethylene glycol (PEG) hydrogel and also on micropatterned poly-caprolactone (PCL) nano-fibrous microwells, which were assembled by photo-patterning of PEG hydrogel in the presence of an electrospun PCL nano-fibrous scaffold. Results showed that a higher level of salivary epithelial markers, tight junction proteins, E-cadherin, and F-actin were expressed in PCL nano-fibrous microwells.²⁸ In another study, PEG hydrogels were suggested as more suitable scaffolds for SG regeneration, while with the introduction of the appropriate growth factors and extracellular matrix (ECM) proteins, they control cell behavior and additionally their stiffness is controlled by the amount of PEG macromeres.²⁹ “Ideal matrix stiffness” is an important characteristic, and high stiffness is undesirable in tissue regeneration.³⁰ The basic problem with polymers is the possibility of inflammation due to their degradation products. Moreover, a silk fibroin scaffold with primary SG epithelial cells from rat major SGs has been established. The cells expressed acinar characteristics and produced amylase in vitro. Thus, silk fibroin is also a promising scaffold for SG regeneration as it is a natural product, biocompatible, and biodegradable with a wide range of elasticities.³¹ Another biocompatible and biodegradable scaffold is a 3D scaffold based on HA, which is abundant in the human body. A 3D HA-based hydrogel scaffold leads to the expression of CD44 and CD168 (RHAMM) markers from human SCs in rodent hosts.³²

Last but not least, a separate reference should be made to nano-fiber scaffolds, which need to be studied further for their establishment in tissue engineering. Nano-fiber and microfiber scaffolds can be created from electrospinning polymers such as poly-l-lactic-co-glycolic acid (PLGA) so that they can mimic the architecture of the basement membrane that surrounds the acinar and epithelial cells. Their ability to stimulate embryonic branching morphogenesis is confirmed and are also considered as the scaffolds that promote the most in vivo like cell morphology, in contrary with microfiber scaffolds.³³ Nano-fiber scaffolds can also be coupled with other substrates like chitosan as mentioned previously. Electrospun nano-fibers are preferred because their micromechanical characteristics (such as diameter, shape, strength, or composition of fibers) can be easily regulated and they can also carry signaling molecules (genes and drugs), which are necessary for differentiation guidance.³⁴

3D bio-printing has also been a technological breakthrough in medicine, which enables the exact display of human organs into 3D structures. Designs are fabricated using the computer-aided design (CAD) software and images are obtained via computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), and 3D printers use this data to produce 3D volumetric structures through a layer-by-layer deposition of materials.³⁵ Inkjet printers, laser-based printers, and micro-extrusion printers are the most commonly used 3D printers, and synthetic polymers, ceramics, and polymeric hydrogels are the most common printable materials.³⁵ Considering their biocompatibility and tenability, polymeric hydrogels are suggested as the best materials for tissue printing.³⁶ Therefore, 3D-printed scaffolds could be designed for each patient and could be excellent for tissue regeneration due to their ability to mimic anatomical features in great detail. Concerning dentistry, attempts have been made to use this technique for the regeneration of periodontal complex, bone, and cartilage in the craniofacial area and for whole teeth regeneration,³⁷ but examples of trials for SG regeneration through 3D printing are still lacking in the literature. It is also remarkable that cells and growth factors are not combined in the polymer mixture as they are susceptible to the high temperatures during the bio-printing process.^35,38 Conclusively, 3D printing is a challenge in tissue regeneration and we are still far from the creation of functional organs in the laboratory. However, 3D printing has a great scope in the future and will become feasible for clinical transplantation purposes. Despite all these findings, the ideal scaffold for in vivo SG regeneration has not yet been established, and further studies are needed.

EXPERIMENTS, ACCOMPLISHMENTS, AND THERAPY POTENTIALS

PANCREAS: A GLAND OF GREAT SIGNIFICANCE, QUITE SIMILAR TO SGS

The pancreas is a gland that produces both hormones and enzymes, playing a crucial role not only in nutritional balance but also in reserving energy for cellular activities. It includes three types of cells: acinar, ductal, and endocrine cells.

There have been numerous efforts to define the areas which home the SCs and to highlight their possible contribution in therapy. In contrast to other organs, the pancreatic SCs are rare in postnatal tissues. Specifically, the replacement of β-cells depends mainly on the expansion of pre-existing rather than differentiation of SCs.⁶¹ Nevertheless, when the pancreas is injured through a duct ligation experiment, a neogenesis of β-cells was detected from Neurog3-expressing islet progenitors.⁶² It is thought that pancreatic ducts⁶³ and pancreatic duct glands (PDGs)⁶⁴ contain committed precursors, which have confined proliferative capabilities. Recently, adult SC populations have been found in the biliary tree both for the pancreas and the liver. These discrete areas of SCs are called niches. Niches consist of peribiliary glands (PBGs), which contain biliary tree SCs (hBTSCs). These cells are precursors to pancreatic SCs (hPSCs).⁶⁵

Regarding possible regenerative potential using SCs derived from the oral cavity, Govindasamy et al. used human dental pulp SCs (DPSCs) to differentiate them into β-cells.⁶⁶ They were in vitro functioned and secrete insulin and C-peptide in a glucose-dependent manner.⁶⁶ SGs and pancreas developmentally originate from the foregut. This might explain the phenotypic similarities and analogous properties of these two functionally different cell populations. Apart from their similar origin, exocrine glands including the pancreas, the liver, and SGs develop via branching morphogenesis. During morphogenesis, each epithelium interacts with gland’s mesenchyme directing gland’s branching morphogenesis. Even though they express different SC markers (embryonic and adult), they could differentiate into ectoderm-, mesoderm-, and endoderm-derived cells.⁶⁷ As exocrine glands, both SGs and the pancreas have glandular SCs (GSCs), which can differentiate into multiple cell types and have plasticity.⁶⁷ Hepatocyte and pancreatic islet-like cells were observed when salivary gland progenitor-1 (SGP-1) cells were cultured on type-I collagen dishes. SGP-1 was obtained after the ligation of the submandibular gland.⁶⁸ The same research team succeeded in differentiating human SG progenitor cells in spherical cultures into NKX6.1 and neurogenin-3 expressing cells which are expressed at the early islet stage.⁶⁹ Insulin and albumin were expressed after differentiation of swine SGP cells in spherical culture, suggesting that they could be used in endodermal regeneration. SGSCs formed pancreatic clusters when they were cultured with activin A, exendin-4, and retinoic acid. They express Pdx1, pan polypeptide, and neurogenin-3 which are pancreatic cell markers.⁷⁰ Differentiation of mouse SG-derived progenitors (mSGPs) into a pancreatic endocrine lineage was successful, when cultured with glucagon-like peptide-1 (GLP-1)⁴⁰ (Fig. 3 and Table 2).

LIVER: AN APPROACH FOR HEPATIC DIFFERENTIATION

Liver’s essential metabolic, endocrine, and exocrine functions are of vital importance for the preservation of life. It regulates blood composition by modulating and releasing glucagon as per needs; processes hemoglobin for iron storage; and produces cholesterol, albumin, clotting factors, and a wide variety of other important proteins. It consists of two groups of cells; parenchymal hepatocytes and biliary epithelial cells, and non-parenchymal cells such as Kupffer cells, stromal cells and stellate cells of mesodermal origin. It is worth mentioning that in case of injury and mass loss, liver possesses the admirable capacity of regeneration which is accomplished through replication of mature adult hepatocytes and other hepatic cell types.⁷¹

Stem/progenitor cells present in the liver have been thoroughly investigated through several researches and in vivo experiments. The fact that hepatocytes are capable of achieving a significant clonal expansion suggests that they themselves could possibly consist of hepatic SCs. However, studies have shown that in case of a severe liver injury, a potential SC population located within the intrahepatic biliary tree is activated and gives rise to a bio-potential cell population called the “oval cells” that can differentiate into hepatocytes. Oval cell populations are positive for hepatocyte and bile ductal cell markers such as albumin, fetoprotein alpha-fetoprotein (AFP), cytokeratin 19, and other bile duct antigens and also contain hematopoietic cell surface antigen-positive cells. Liver regeneration does not include the proliferation of these types of SCs. On the contrary, oval cells are noticed during progenitor-dependent liver regeneration, which has been shown under different experimental conditions.⁷² One representative protocol⁷³ combined the administration of 2-acetylaminofluorene (2-AAF) with partial hepatectomy and results showed that under these circumstances, 2-AAF suppressed hepatocytes proliferation and oval cells appeared in the peri-portal area before regeneration. It has also been suggested that when all other means of regeneration fail, a third population of hematopoietic SCs derived from bone marrow can differentiate into hepatocytes or oval cells and rescue the liver.⁷⁴

Identification and isolation of hepatic SCs are assisted by several stem/progenitor markers including epithelial cell adhesion molecule (EpCAM), CD133 (prominin), CD44 (hyaluronan receptors), ALDH hedgehog proteins, NCAM and CXCR4. Hepatic SCs can also be culture selected with a hormonally defined medium, called Kubota’s medium, that supports hepatic progenitors but not mature cells.⁷⁵ Biomatrix scaffolds that contain extracellular matrix components, matrix-bound cytokines, and growth factors could provide the necessary chemical signals for a stable scaffolding that can be used for inducing hepatic SCs into liver fetes or just for the maintenance of mature hepatic cells for many weeks (%3E;8 weeks) without loss of their viability.⁷⁶

Several researches have been conducted to prove whether or not, tissue SCs from the liver could undergo trans-differentiation to cells of other tissues and vice versa. For example, submandibular SGs originate from the endoderm and the ectoderm which indicates the possible presence of endodermal progenitor cells. This case was investigated by Okimura et al. in their experiment when they cultured cells obtained from the ligated SG and identified colonies of epithelium-like cells. Then they singled out and purified the cells by limited dilution, and one of the cells designated SGP-1 was used for further experiments. The hematopoietic SC marker CD34 and hepatic oval cell markers such as albumin, AFP, and cytokeratin-19 were all negative, but when SGP-1 cells were transplanted into the liver via the portal vein, these cells were integrated into the hepatic trabecule and produced the albumin. When SGP-1 cells were cultured with collagen type 1, they were differentiated into hepatic and pancreatic lineages. In conclusion, the multipotent progenitor cells isolated from the rat SG have characteristics of tissue SCs and can differentiate into cells of endodermal lineages.⁶⁸ Recently, a study on minor SG cells proved their ability to express liver-associated markers (ALB, CYP3A4, AAT, and CK18) in vitro, which also improved severe acute liver damage in severe-combined immunodeficiency (SCID) mice when injected in vivo.⁴ It would be a great step to combine minor SG cells with those organs cells for the purpose of tissue regeneration (Table 3).

CONCLUSION

SC research expands the knowledge concerning therapeutic opportunities for SG hypofunction, and even other glandular organ failures. An autologous treatment using patient’s own SCs may provide an easy, low-cost, and a less complicated solution. Also, such approaches will exhibit ideal tissue tolerance and no local inflammation following transplantation. SCs without regard to their tissue of origin universally express great potential to regenerate, opening a new door to individualized therapy. Especially, the prospect of using SCs derived from minor SGs could be even less traumatic, and an efficient source of SC therapy in the near future.

However, SC therapeutic application is currently limited by several issues including immunological host vs graft reactions which needs to be considered. Their capability to differentiate into various cell lines makes it difficult to reach a functional tissue differentiation. Long-term possibility of carcinogenesis, somatic mutations, epigenetic defects induced during reprogramming, and their lack of stability in vivo are significant limitations of SCs, especially embryonic and pluripotent SCs, which prevents from using them in therapies.^55,56 Further clinical studies are needed, to overcome these barriers and to attain the promises of SC-based regenerative medicine. Besides, surmounting these limitations will give us the opportunity to understand in detail not only disease’s pathogenesis but also the complexity of embryogenesis. SCs hold a tremendous potential, so reducing the limitations can lead to unlimited possibilities. It would be even exciting and optimistic for tissues such as SGs, the liver, the pancreas, and even thyroid and the kidney to exchange SCs and provide each other regeneration.

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