Salivary Gland Stem Cells and Tissue Regeneration: An Update on Possible Therapeutic Application
1–3,5,8Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece
4Department of Prosthodontics and Implantology-Tissue Regeneration Unit, School of Dentistry, Aristotle University of Thessaloniki, Greece
6Department of Oral Medicine and Radiology, MNR Dental College and Hospital, Sangareddy, Telangana, India
7Department of Maxillofacial Surgery and Diagnostic Sciences, Division of Oral Pathology, College of Dentistry, Jazan University, Jazan, Kingdom of Saudi Arabia
Corresponding Author: Prashanth Panta, Department of Oral Medicine and Radiology, MNR Dental College and Hospital, Sangareddy, Telangana, India, Phone: +91 9701806830, e-mail: firstname.lastname@example.org
How to cite this article Mitroulia A, Gavriiloglou M, et al. Salivary Gland Stem Cells and Tissue Regeneration: An Update on Possible Therapeutic Application. J Contemp Dent Pract 2019;20(8):978–986.
Source of support: Nil
Conflict of interest: None
The aim of this review is to combine literature and experimental data concerning the impact of salivary gland (SG) stem cells (SCs) and their therapeutic prospects in tissue regeneration. So far, SCs were isolated from human and rodent major and minor SGs that enabled their regeneration. Several scaffolds were also combined with “SCs” and different “proteins” to achieve guided differentiation, although none have been proven as ideal. A new aspect of SC therapy aims to establish a vice versa relationship between SG and other ecto- or endodermal organs such as the pancreas, liver, kidneys, and thyroid. SC therapy could be a cheap and simple, non-traumatic, and individualized therapy for medically challenging cases like xerostomia and major organ failures. Functional improvement has been achieved in these organs, but till date, the whole organ in vivo regeneration was not achieved. Concerns about malignant formations and possible failures are yet to be resolved. In this review article, we highlight the basic embryology of SGs, existence of SG SCs with a detailed exploration of various cellular markers, scaffolds for tissue engineering, and, in the later part, cover potential therapeutic applications with a special focus on the pancreas and liver.
Keywords: Salivary gland stem cells, Stem cell therapy, Tissue regeneration.
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
|Marker||Role||In vitro* differentiation||In vivo function||Location|
|CD24||Sialoglycoproteinic cell adhesion molecule acting expressed in progenitor cells||Human**||Yes (mouse)||Cell membrane|
|CD34||Marker expressed in early lympho-hematopoietic stem and progenitor cells, and embryonic fibroblasts||Human**||No||Cell membrane|
|CD117/CKIT||Tyrosine kinase receptor expressed in endothelial, epithelial, and endocrine cells during development||Mouse||Yes (mouse)||Cell membrane|
|CD44||Leukocyte common antigen (LCA), hematopoietic marker||Human*||No||Cell membrane|
|CD271/NGFR||Low-affinity nerve growth factor receptor, SC marker||Human**||No||Cell membrane|
|Nanog||Embryonic SC marker||Human**||No||Intracellular|
|Aquaporin-1||Water channel’s molecule expressed in developed SGs||Mouse**||No||Cell membrane|
|Oct 3/4||Embryonic SC marker||Human**||No||Intracellular|
|Stro-1||Early MSC marker||Human**||No||Cell membrane|
|Nestin||SC marker and marker of proliferating and migrating cells||Human**||No||Intracellular|
|CD90/Thy1||Cell adhesion molecule, expressed in hematopoietic and adult hepatic stem/progenitor cells||Human**||No||Cell membrane|
|CD81/TAPA||Tetraspanina molecule related to signal transduction, cell development, growth, and motility||Human**||No||Cell membrane|
|CD105/endoglin||Cytokine which acts as a part of TGF-a receptor complex and participates in cell proliferation, differentiation, and migration||Human**||No||Cell membrane|
|CD49f/a6 integrin||Cell adhesion molecule which mediates intercellular and cell-matrix interactions and cell migration||Human**||Yes(mouse)||Cell membrane|
|SSEA-3||Embryonic stem cell marker||Human**||No||Cell membrane|
|CD146/MUC18||Perivascular marker associated to multipotency and MSC multilineage differentiation potential||Human**||No||Cell membrane|
|ALDH||Enzymes involved in aldehyde detoxification and in the metabolism of retinoic acid, biogenic amines, and neurotransmitters||Human**||No||Cell membrane|
|Ascl-3||A transcriptional factor localized primarily to ductal cells||Human**||No||Cell membrane|
|TRA-1-60||Embryonic SC marker related to mesenchymal, hepatic, cardiac, pancreatic, and neural cell||Human**||No||Cell membrane|
|CD298/Na-K-ATPase B3||Component of the cell membrane sodium pumps||Human**||No||Cell membrane|
** Salispheres culture method
*** Monolayer culture method
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
Transplantation in Murine
The use of SCs as a treatment option for hyposalivation has been an ambitious plan for over a decade. The first attempt was made in 2004, but SCs failed to differentiate into saliva producing acinar cells or myoepithelial cells in rat SGs, 4 weeks after transplantation.39 This study established the feasibility of a successful and biocompatible transplantation with the simultaneous cell adhesion.39 On the contrary, the detection of Sca-1/c-kit+ cells’ population from mice SGs and their differentiation into hepatic cells when cultured in matrigel and into a pancreatic lineage with the presence of glycagon-like-peptide 1 (GLP-1) was a breakthrough of the same year.40 Lombaert et al.18 later cultured in vitro c-kit+ SCs that originated from murine submandibular glands and injected into severely damaged irradiated murine SGs. These cells managed to express amylase and mucin, while rescuing the glands hypofunction at some point.18 Based on this ability of c-kit cells to repair damaged SGs, investigators have tried to translate this in humans. Another study proved that c-kit-positive cells could also be isolated from human exocrine ducts, organize in salispheres, and express amylase and mucin, promising an equivalent restoring ability.41 Transplantations of SCs from various tissues are currently being conducted and evaluated for efficacy and safety.
Adipose-derived cells (ADSCs) were isolated and transplanted into mice and accomplished to differentiate into endothelial cells, which improved blood flow, an important factor for saliva flow. ADSCs were considered as cells with strong potential of restoring many parts of SGs without infiltrating the surrounding tissue.42
In a different model, during the same time, bone marrow cells were examined as a possible source of therapeutic SCs and were engrafted into mice and rats. The mouse model reacted better than the rat model as bone marrow SCs accomplished functional regeneration of the SGs. It should also be noted that acinar-like cells used in the same study showed better-restoring capability than bone marrow cells.43 Lately, trans-differentiation was observed in acinar cells. These cells have been found to improve saliva secretion, reduce apoptosis, and help microvessel regeneration in mice.54
As progress in rat and mice models is accomplished, studies begin to create human SCs cultures and assess their potential. A novel in vivo model of a tissue-engineered SG from human SG progenitor cells was investigated with the culturing of human SG cells in HA hydrogels. Injections of human SCs into rodents led to saliva production and regeneration of irradiated murine SGs, for the first time due to a self-regenerating kit+ cells population.44 At the same time, SG tissues were characterized by an activation of the Wnt pathway, which is crucial for the self-renewal in vitro.44
Nevertheless, first-in-man mesenchymal SCs use for radiation-induced xerostomia (MESRIX) was conducted in 2017, including a double-blinding clinical trial with autologous adipose-derived MSC therapy or placebo therapy (no results yet). Evidence still needs to be evaluated and further investigated as this study is established as the first clinical trial in humans intending to estimate feasibility, efficacy, and safety of such project.45 These studies point out at the possible application of this method especially for xerostomia induced by radiation exposure in head and neck cancer patients.
Autologous SCs Transplant in SGs
Cell-based therapy26 can also be used, when glands are partially or globally injured, they can be restored by transplanting cells at the end of the treatment. These SCs are isolated and preserved in cultures before treatment.18 However, due to aging, patients contain less number of SCs. To overcome this, recent studies focus on the use of growth factors51 or the ALDH3 activator20 so as to increase the number of kit+ cells ex vivo. Cryopreservation can be a therapy solution as Neumann et al. developed a SC banking model where SG CD49f+ CD29+ cells were preserved up to 3 years without any distortion.52
Regeneration Methods/Gene Therapy
Therapy potentials of xerostomia aim for SG regeneration and their functional restoration. However, so far, no such therapy was found. Current, innovative therapies include gene therapy which is a method that uses transferred genes into cells so as to treat a disease or correct a cellular dysfunction. Promising results have been reported in a clinical trial, where 5 of 11 enrolled subjects had an increased saliva flow rate over the first 42 days. Their irradiated parotid glands was delivered AdhAQP1, which is the first-generation serotype 5 adenoviral (Ad5) administering the hAQP1 gene.46 Remarkably, all five subjects reported higher levels of parotic saliva flow rates even 5 years later.47 Another study used a hybrid serotype S adenovirus vector as a transferring means for human KGF gene.48 Saliva flow was increased and acinar cell proliferation occurred. In addition, concurrent transient activation of Wnt/β-catenin pathway in male mice preserved SG function by inhibition of apoptosis and preservation of functional SCs.49
Another alternative gene therapy is gene activation. ALDA-89 is a selective ALDH3 activator, which enriched kit+/CD90+ SC population. Higher levels of ALDH3 were examined in those cells in comparison with those in non-SCs. This led to increased proliferation and preservation of SG SCs.20 The improvement of saliva secretion could be achieved by retrograde injection of siRNA-coated nano-particles, which ensures direct accessibility to gland parenchyma. This method targeted a pro-apoptotic Pkcδ gene, which was silenced and apoptosis was prevented.50
Bioengineering of SG Germ
The glandular function could be restored with the implantation of a glandular structure bioengineered in vitro. Recently, a bioengineered SG germ was transplanted into adult mice, resulting in the regeneration of SG function.53 Morphogenesis was successful through epithelial and mesenchymal interactions. The existing ducts and the transplanted SGs were reconnected. Specifically, the transplanted submandibular gland was capable for saliva secretion, protection of the oral cavity from bacteria, and restoration of normal swallowing.
Tissue Engineering with Scaffolds
A biocompatible scaffold that simulates gland’s microenvironment and pluripotent cells that preserve salivary markers consists of tissue engineering. Embryonic SCs55 and induced pluripotent SCs56 can be a treatment option, as they are stable and without oncogenic potential. The cells are seeded in a scaffold so as to recreate an artificial gland.57,58 A hydrogel culture system was capable of maintaining 3D salivary spheroid structures and for acini-like structures in vitro, whereas it retained viability, when transplanted into rats. In addition, poly lactic-glycolic acid is a promising scaffold, as it supports branching of fetal SMGs.59 Moreover, a study used SGs cells derived from a 3D coculture of mouse ES cells and human SG-derived fibroblasts. In vitro, the cells are supposed to have the neogenetic ability, though it is still unclear whether they maintain stability in vivo.55 Another research approach is to use the HSY cell line, which are neoplastic epithelial cells that are derived from transplanted human parotid gland adenocarcinoma to athymic mice tumors, for bioengineering therapy. These cells are morphologically similar to intercalated duct cells, which are a reservoir of progenitor SCs.60
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
|Endoderm Induction markers*||CD184, CD117, EPCAM, CD49e, CD51, CD141, and CD238|
|Pancreatic progenitor marker||Sox9|
|Mature islet cell surface markers||FXYD2, tetraspanin7 (TSPAN7), Tmem27, δ-, α- and β-cell surface antibodies, and pan-islet antibodies|
|Endocrine surface markers||DDR1, DNER, Pdx1, MafA, Ngn3, NeuroD, ΝΚΧ6.1, and Ptf1a|
|Surface markers of hormone-positive cells||SEZ6L2, DISP2, LRP11, and CD24|
|Acinar epithelial cell markers||NKX6.1 and Ptf1a|
|Ductal epithelial cell markers||CD133 and CD149f|
* None of these is exclusively specific for endoderm lineage77
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
|CD24||Sialoglycoprotein adhesion molecule expressed in progenitor cells||Surface markers of hormone-positive cells77||–||Pluripotency marker during SC differentiation|
|CD34||CD34+ cells human parotid, sublingual and submandibular glands78||–||CD34+ cells from human fetal liver express biliary epithelial markers CK7, CK8, and CK1879||Cell surface glycoprotein|
|General marker for progenitor cells|
|CD44||Human SCs80||Pancreatic mesenchymal SCs (pMSC) that generate at least two germ layer cells, including endoderm-derived cells, but still no convincing evidence of pancreatic lineage cells81||–||Hematopoietic marker|
|CD90 (Thy1)||Human SCs80||Pancreatic mesenchymal SCs (pMSC)81||Human hepatic progenitor cells (HPCs)82||Cell adhesive molecule|
|Sox-2||Regulates acinar cell development in SG83||*||–||Transcriptional regulator in pluripotent SCs|
|CD133||Ductal SC expression in humans84||Potential reservoir of side population of adult stem/progenitor cells that express CD13385||–||Transmembrane glycoprotein and SC marker|
* Undetermined role
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).
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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