Med Lasers 2024; 13(1): 19-24  https://doi.org/10.25289/ML.24.010
In vitro stem cell differentiation for salivary gland regeneration: a comprehensive review
Hin Kei Kim1, Seung Hoon Woo2
1School of Medical Laser, Dankook University, Cheonan, Republic of Korea
2Department of Otolaryngology-Head and Neck Surgery, Dankook University College of Medicine, Cheonan, Republic of Korea
Correspondence to: Seung Hoon Woo
E-mail: lesaby@hanmail.net
ORCID: https://orcid.org/0000-0001-7560-1140
Received: March 13, 2024; Accepted: March 20, 2024; Published online: March 30, 2024.
© Korean Society for Laser Medicine and Surgery. All rights reserved.

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Salivary gland dysfunction significantly affects oral health and the quality of life, necessitating the development of effective regenerative therapies. In recent years, in vitro stem cell differentiation has emerged as a promising approach for salivary gland regeneration. This review provides a comprehensive overview of the current state of research in this field and discusses various types of stem cells, differentiation protocols, and the challenges and opportunities for translation to clinical practice. Additionally, we explore the potential of stem cell-based therapies which may revolutionize salivary gland regeneration and improve patient outcomes in the future.
Keywords: Stem cells; Salivary glands; Xerostomia; Treatment; Regeneration
INTRODUCTION

The salivary gland (SG) comprises the secretory acini and ductal parts, together responsible for the production and transportation of saliva. Saliva acts as a lubricant, antimicrobial and digestive agent, thus being responsible for oral and general health. Over 90% of saliva is produced by the three major SGs, namely parotid glands, sublingual glands and submandibular glands [1], and a possible fourth one recently identified with the proposed name tubarial glands [2]. Moreover, there are some minor SGs in the oral lining.

Impairment of SGs would reduce saliva production, perceived by patients as mouth dryness, a clinical condition named xerostomia. Xerostomia can be caused by medication, autoimmune attacks for people with Sjögren’s syndrome, radiation therapy for head and neck cancer patients, and aging [3,4]. Xerostomia patients suffer from not only the sensation of mouth dryness, but also difficulty in speaking and swallowing, and oral diseases like dental caries and infection [1].

The most common treatments for SG dysfunction are symptom management using artificial saliva and salivary stimulants, which are palliative [4]. Other more controversial and developing treatment methods include immune response modulation for Sjögren’s syndrome patients, stem cell therapy, preventive medication prior to radiotherapy and gene therapy [5].

Stem cell-based approaches hold great promise for SG regeneration due to their unique ability to differentiate into various cell types and promote tissue repair. Previous animal studies on engrafting mesenchymal stromal cells (MSCs) into damaged SG showed increased salivary flow rate and SG mass [6]. Meanwhile, Lin et al. (2011) [7] have shown a more desirable effect using MSC-derived acinar-like cells for treatment compared to using MSC directly. This suggests the therapeutic potential of cell therapy using progenitor or SG cells for SG regeneration, thus the importance of developing ways to generate SG-like cells or even functional SG cells in vitro. In this review, we would like to summarize current knowledge on stem cell differentiation into SG cells, focusing on in vitro experiments.

METHOD

The PubMed database is used to search for papers from the last 10 years using the keywords “stem cell,” “differentiation,” and “salivary gland.” Total 212 results are generated, among which 14 most relevant papers are selected. This 11 are original research articles and 3 of them are review articles.

RESULT

Table 1 [8-21] summarizes the stem cell sources, differentiation methods and SG protein markers in the selected papers, arranged in the order of year published.

Table 1 . Experimental summary of 14 salivary gland (SG) differentiation studies

Stem cell sourceDifferentiation methodSG protein markerResultReference
Murine SMGCultured as salispheresCK7, CK14, PASThere were stem cell present in the SMG isolation and the stem cell was able to differentiate into acinar cells, also indicated by an increase in amylase expressionLombaert et al., 2008 [8]
Human MSCCoculture with human SGCLDN1,2,3,4, occludin, JAM-A, zonula occludens-1, AQP5, AMY1, E-cad20%-40% of MSC expressed tight junction proteins and other epithelial markers. The cellular structure is comparable to human SG cells with tight junction structures and secretory granules. The amounts of AMY1 detected were comparable to that of the human SG control cultureMaria and Tran, 2011 [9]
Mouse ESCCoculturing with human SG-derived fibroblastsAMY, AQP5, CK, bFGF, NGFThe cocultured cells expressed SG markers and reconstituted SG structure in the 3D culture systemKawakami et al., 2013 [10]
BM-MSCCocultured with primary salivary epithelial cellsAMY1, M3R, AQP5, CK19Cocultured cells resembled salivary epithelial cells and were confirmed by the expression of SG epithelial markers. In addition, ANKRD56, HMG20B, and TCF3 were identified as regulatory factors for MSC transdifferentiationPark et al., 2014 [11]
A-MSC1. Cocultured with acinar cells
2. Cultured in an acinar-conditioned medium
AMY1, AQP5Both differentiation methods showed expression in acinar markers. However, the coculture system has lower senescence than the acinar-conditioned medium culture groupLee et al., 2015 [12]
Embryonic SMGCoculture with iPSCPSP, AMY, E-cad, AQP5Cocultured cells have better-developed epithelial structures and less undifferentiated markers than monoculture of SMGOno et al., 2015 [13]
A-MSCCoculture with human SG fibroblastAMY, bFGF, NGFCocultured cells expressed SG markers and formed glandular structures in the 3D culture systemKawakami et al., 2016 [14]
Mouse BM-MSCCocultured with primary mouse SG cellsAMY1, M3R, AQP5With high-throughput proteomics, induction of PTF1α, MIST1, ASCL3 were identifiedPark et al., 2017 [15]
Mouse ESCRecombinant adenovirus infection of SOX9 and FOXC1Pan-CK, K18, AQP5, α-SMATranscription factors SOX9 and FOXC1 were identified to be responsible for the differentiation of oral ectoderm into SG rudimentTanaka et al., 2018 [16]
Mouse BM-MSCCocultured with primary salivary epithelial cellsAMY1, M3R, AQP5By high-throughput liquid chromatography with tandem mass spectrometry, protein IGFBP7, CYR61, AGRN, LAMB2, FSTL1, FN1 were identified as potential contributors to MSC transdifferentiation into SGMona et al., 2020 [17]
Mouse ESCBMP4 treatment, followed by retinoic acid and bFGF, then FGF10PITX1, E-cad, SOX9, K5, K19Treatment of retinoic acid and FGF10 promoted the differentiation of stem cell into SG placodes, recapitulating the morphogenetic stages in the fetusZhang et al., 2022 [18]
Urine-derived iPSCTransfected with IRF6 and culture in parotid conditioned mediumAMYIRF6 group and only conditioned medium group both showed increase in amylase expression, with the IRF6 group showing higher expression. Also, transcriptome sequencing showed that SG related genes HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1, and LUM were upregulatedMeng et al., 2022 [19]
BM-MSCTreated with decellularized rat SG-extracellular matrixAMY, CLDN3 and 10, AQP5, MIST1, K14Treated cells expressed AMY, tight junction proteins (CLDN3 and 10), acinar marker (AQP5 and MIST1) and ductal marker (K14). They also formed secretory granules, which were morphologically similar to SMGTran et al., 2022 [20]
Human PSCEmbryoid bodies formation, followed by treatment of BMP4, retinoic acid, and CHIR99021SOX9, K5, K19, CD24, α-SMAProgenitor markers of developing SG SOX9, K5, K19 were expressed. CD24 and α-SMA positive cells, which can restore SG function, were presentZhang et al., 2023 [21]

Stem cell source: SMG, submandibular gland; MSC, mesenchymal stromal cell; ESC, embryonic stem cell; BM-MSC, bone marrow derived-MSC; A-MSC, adipose-derived MSC; iPSC, induced pluripotent stem cell; PSC, pluripotent stem cell.

Differentiation method: SOX, SRY-box transcription factor; FOXC1, forkhead box C1 protein; BMP4, bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; FGF10, fibroblast growth factor 10; IRF6, interferon regulatory factor 6; CHIR99021, glycogen synthase kinase 3 inhibitor.

SG protein marker: CK, cytokeratin; PAS, Periodic acid Schiff; CLDN, claudin; JAM-A, junctional adhesion molecule A; AQP5, aquaporin-5; AMY, α-amylase; E-cad, E-cadherin; NGF, nerve growth factor; M3R, muscarinic-type-3-receptor; PSP, parotid secretory protein; K, keratin; α-SMA, α-smooth muscle actin; PITX1, pituitary homebox 1; MIST1, muscle, intestine and stomach expression-1; CD24, cluster of differentiation 24.

Result: ANKRD56, ankyrin-repeat-domain-containing-protein 56; HMG20B, high-mobility-group-protein 20B; TCF3, transcription factors E2a; PTF1α, pancreas-specific transcription factor 1α; ASCL3, achaete-scute complex homolog 3; IGFBP7, insulin-like growth factor binding protein-7; CYR61, cysteine-rich angiogenic inducer 61; AGRN, agrin; LAMB2, laminin beta 2; FSTL1, follistatin-like 1; FN1, fibronectin 1.



Types of stem cells

Several stem cell sources are used, including primary SG isolated from mice, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and MSC (Fig. 1). ESCs offer pluripotency and the ability to differentiate into any cell type in the body, including SG epithelial cells. However, ethical concerns and immune rejection limit their clinical applicability. Induced pluripotent iPSCs, derived from adult somatic cells through reprogramming, overcome these limitations and have been successfully differentiated into SG-like cells in vitro.

Figure 1. Stem cell sources that have been used for salivary gland differentiation. iPSC, induced pluripotent stem cell; MSC, mesenchymal stromal cell.

In recent years, MSCs have been of great interest to research because of their trans-differentiation potential and avoided ethical concerns about using ESC. MSCs are also well characterized by the cell surface markers including cluster of differentiation 73 (CD73), CD90, and CD105, but the absence of CD11b, CD14, CD19, CD34, CD45, CD79a, and HLA-DR [22]. It is also worth noting that the scientific community has gradually come to regard MSC not as stem cells, thus more scientists have been employing the new name of MSCs.

Differentiation methods

The differentiation methods employed can be categorized into 3 groups, the coculture system, factors, and vector transfection (Fig. 2). A gradual development from culturing SG stem cell, co-culturing other stem cell sources with SG cells, to using factors or vector transfection to induce differentiation can be seen. This is due to the increased understanding of SG development, and more advanced technology available to analyze protein and gene expression on a larger scale.

Figure 2. Differentiation methods.

In addition, several groups used the three-dimensional (3D) culture method. The 3D culture is known to better mimic that of cells in vivo, thus allowing researchers to perform more accurate and advanced experiments [23].

Markers

The markers can be classified into 3 groups, the stem cell markers, SG progenitor markers, and SG markers.

1. Common stem cell markers include c-KIT, Sca-1, Musashi-1, SOX2, NANOG, c-MYC, KLF4, SSEA4, and OCT4 [5,8,13,19].

2. For SG progenitor markers, there are c-KIT, K5, K14, K19, AXIN2, SOX2, SOX9, SOX10, LGR5, CD90, and PITX1 [5,18].

3. For SG markers, they are subdivided into different cell types. Epithelial cells, also known as luminal cells, include the acinar and ductal cells. The acinar cells are secretory cells, which consist of serous and mucous cells, secreting watery serous saliva and mucous respectively [24]. The ductal cells are located along the intercalated ducts, striated duct and downstream. The structure of the SG and markers of different cell types are shown in Fig. 3.

Figure 3. Schematic diagram showing localization of cellular markers in the salivary gland: CK, cytokeratin; K, keratin; AMY1, α-amylase 1; M3R, muscarinic-type-3-receptor; AQP5, aquaporin-5; MIST1, muscle, intestine and stomach expression-1; PAS, Periodic acid Schiff; α-SMA, α-smooth muscle actin; CD24, cluster of differentiation 24.

Application and limitation

The final goal of differentiating SG cells is to provide a cure for xerostomia patients. There have been clinical trials of allogeneic MSC transplantation, but they showed only short-term effect and could not repair damaged cells in the long term [6]. A possible reason would be immune rejection of MSC [25]. Researchers may consider exploring the effectiveness of autologous MSC for the regeneration of SGs.

The current understanding of underlying mechanisms in stem cell differentiation into SG cells is very limited. Signaling pathways involved in SG development, including retinoic acid signaling, fibroblast growth factor signaling, epidermal growth factor receptor family signaling, and Wnt, Hippo, and Notch signaling can be further explored [26].

In addition, a recent study published that about half of the laboratories used stem cells containing cancer phenotype [27]. Regulation of stem cell quality would be a challenge in cell therapy.

Challenges and future directions

Despite significant progress, several challenges remain to be addressed before in vitro stem cell differentiation can be translated into clinical therapies for SG regeneration. These challenges include improving the efficiency and consistency of differentiation protocols, optimizing cell survival and integration following transplantation, and ensuring the safety and long-term efficacy of stem cell-based therapies. Additionally, the development of biomaterials and scaffolds that mimic the native SG microenvironment may enhance the functional maturation and integration of transplanted cells.

CONCLUSION

In vitro stem cell differentiation holds immense potential for SG regeneration and the treatment of xerostomia. By harnessing the regenerative capacity of stem cells and understanding the underlying mechanisms of gland development, researchers are paving the way for innovative therapies that could significantly improve the quality of life for patients with SG dysfunction. Continued interdisciplinary research efforts are essential to overcome remaining challenges and realize the full clinical potential of stem cell-based approaches for SG regeneration.

SUPPLEMENTARY MATERIALS

None.

ACKNOWLEDGMENTS

None.

AUTHOR CONTRIBUTIONS

Conceptualization: SHW, HKK. Data curation: HKK. Formal analysis: HKK. Investigation: HKK. Methodology: HKK. Project administration: SHW. Software: HKK. Validation: SHW. Visualization: HKK. Writing–original draft: HKK. Writing– review & editing: all authors.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

FUNDING

None.

DATA AVAILABILITY

None.

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