Oral Organoids: Progress and Challenges

Abstract

Oral organoids are complex 3-dimensional structures that develop from stem cells or organ-specific progenitors through a process of self-organization and re-create architectures and functionalities similar to in vivo organs and tissues in the oral and maxillofacial region. Recently, striking advancements have been made in the construction and application of oral organoids of the tooth, salivary gland, and tongue. Dental epithelial and mesenchymal cells isolated from tooth germs or derived from pluripotent stem cells could generate tooth germ–like organoids by self-organization in a specific culture system. Tooth organoids can also be constructed based on tissue engineering principles by seeding stem cells on a scaffold with the bioregulatory functions of odontogenic differentiation. Two main approaches have been used to construct salivary gland organoids: 1) incubation of salivary gland–derived stem/progenitor cells in a 3-dimensional culture system to form the structure of the gland through mimicking regenerative processes and 2) inducing of pluripotent stem cells to generate embryonic salivary glands by replicating the development process. Taste bud organoids can be generated by embedding isolated circumvallate papilla tissue in Matrigel with a mixture of growth factors, while lingual epithelial organoids have been constructed using lingual stem cells in a suitable culture system containing specific signaling molecules. These oral organoids usually maintain the main functions and characteristic structures of the corresponding organ to a certain extent. Furthermore, using cells isolated from patients, oral organoids could replicate specific diseases such as maxillofacial tumors and tooth dysplasia. Until now, oral organoids have been applied in the study of mechanisms of tooth development, pathology and regeneration of the salivary gland, and precision therapeutics for tongue cancer. These findings strongly demonstrate that the organoid technique is a novel paradigm for the study of the development, pathology, and regeneration of oral and maxillofacial tissue.

Keywords

tooth regeneration salivary gland tongue stem cells regeneration culture technique

Introduction

An organoid is defined as a complex 3-dimensional (3D) structure developed from somatic cells, adult stem cell/progenitor cells, pluripotent stem cells (PSCs), or specific cell lines and displays architectures and functionalities similar to in vivo organs (Li et al. 2019; Liu et al. 2019). Organoids can give rise to cell lineages similar to the living tissue and maintain stable inheritance of the genome by mimicking cell-cell and cell-matrix interactions (Clevers 2016). They are more representative of in vivo physiology compared to cells cultured in a conventional 2-dimensional (2D) system. Organoids provide a more stable system amenable to extended cultivation and manipulation of niche components, signaling pathways, and genome editing compared with in vivo models (Rossi et al. 2018). As a new technical system and research method, organoids represent an important bridge between 2D cell cultures and in vivo animal models.

Recently, several oral organoids, including tooth germ organoids, salivary gland organoids, lingual epithelium organoids, and taste bud organoids, have been successfully constructed. Oral organoids can be derived from somatic cells, PSCs, or stem/progenitor cells from oral organs. Under specific culture conditions, stem cells spontaneously self-organize into properly differentiated functional cell types, which resemble their in vivo counterparts and recapitulate at least some functions of the oral organ. Recently, oral organoids have been used in the fields of oral organogenesis, disease model construction, and regenerative medicine.

In this review, we summarized the progress in the establishment of oral organoids and discussed the remaining challenges to the application of organoids in the study of oral physiology and disease.

Construction of Oral Organoids

There are 3 essential elements in the construction of oral organoids: cells, scaffold, and construction strategies. In general, cells exhibit an intrinsic ability to assemble into complex structures when placed in a 3D environment in the presence of specific growth factors and small molecules. Scaffolds, although not necessary for the construction of all organoids, play an important role in the construction of many organoids. In the process of oral organoids formation, the suitable sources of stem cells are the essential elements, and the stem cell microenvironment (or niche) offers a key point of control.

Cells

Because of limitations in the availability, expandability, and throughput of tissues needed for somatic cell organoids, they are less widely used than stem cell organoids and are not the focus of this article (Li et al. 2019). In addition to the cells mentioned above, cell lines have also been used as a source of cells in the construction of salivary gland organoids (Athwal and Lombaert 2019).

Stem cells are defined as cells with the capacity to both self-renew and give rise to differentiated cells (Ramalho et al. 2007). Oral organoids have been derived from 2 main types of stem cells: adult stem cells (ASCs)/progenitor cells and PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Both approaches are based on stem cells’ capacity for multipotent differentiation and self-renewal. In recent years, cocktails of growth factors and small molecules that mimic the niche of various organ stem cells during organogenesis have been developed rapidly. When cultured in a suitable microenvironment, ASCs and PSCs display strong abilities to differentiate into specific lineages and self-organize into structures of certain organs.

Adult Stem Cells/Progenitor Cells

ASCs, also known as tissue stem cells, are defined as cells with intrinsic abilities to self-renew and differentiate into the cell types present in tissues while retaining genomic stability (Huch et al. 2015). ASCs derived from oral tissues have been used in the construction of oral organoids. Currently, tooth germ–derived dental epithelial (DE) and dental mesenchymal (DM) cells have been used in the construction of tooth germ organoids. In conventional tooth germ culture medium (Dulbecco’s modified Eagle’s medium [DMEM]/F12 supplemented with 1% Pen-Strep, 10% fetal bovine serum [FBS], and 0.05 mM ascorbic acid), DE and DM cells can differentiate into ameloblasts and odontoblasts, respectively (Nakao et al. 2007; Smith et al. 2017). Dental epithelial and mesenchymal cells create cellular interactions through their outstanding capacity for self-organization. However, it is difficult to obtain human embryonic cells for tooth regeneration. Dental pulp stem cells (DPSCs) can be easily obtained as ASCs in dental pulp tissues and have odontogenic ability (Sharpe 2016). DPSCs have been used to mimic dental mesenchyme and construct tooth germ organoids (Rosowski et al. 2019). In the construction of salivary gland organoids, stem/progenitor cells with self-organization and self-renewal ability are usually derived from embryonic salivary glands and postnatal ones (Hosseini et al. 2019; Sui et al. 2020). The SIMS cell line, which includes immortalized adult mouse submandibular salivary gland cells, also can differentiate into unique populations of acinar, myoepithelial, and duct cells under various 3D differentiation conditions (Athwal and Lombaert 2019). In the construction of taste bud organoids, Lgr5⁺ stem cells from the circumvallate papillae have been shown to generate organoids containing differentiated taste cells (Aihara et al. 2015). Nevertheless, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not yet been identified. Recently, Bmi1-positive cells isolated from the basal layer of the lingual epithelium have been used to establish lingual epithelium organoids, which are considered to be one type of lingual epithelial stem cells (LESCs) (Hisha et al. 2016).

The advantage of ASCs is that they are tissue specific and prone to differentiate into certain cell types. Therefore, ASCs can spontaneously generate specific organoids in culture conditions mimicking the molecular environment in adult tissue (Bartfeld and Clevers 2017). However, this method is hindered by the limited source of ASCs from oral tissue.

Pluripotent Stem Cells

The use of PSCs in the establishment of oral organoids has many outstanding advantages, including excellent multipotent differentiation and self-renewal capabilities. Recent reports have shown that mouse iPSCs could differentiate into ameloblastin-expressing DE cells, when co-cultured with rat dental epithelial cells (SF2-24) or DE tissue, and odontogenic mesenchymal cells through neural crest–like cells, when cocultured with DM cells or tissue (Kim et al. 2019). However, the induction of PSCs through coculture is limited due to the lack of suitable sources of tooth germs. Alternatively, an induction medium containing key signal molecules from tooth germs could be applied to solve the problem. For example, the addition of BMP4, retinoic acid, and N2 to serum-free keratinocyte synthetic medium could induce mouse iPSCs to differentiate into DE cells (Abdullah et al. 2019). A neural induction medium consisting of a 1:1 ratio of DMEM/F12 and neurobasal medium supplemented with N2, B27, epidermal growth factor (EGF), and fibroblast growth factor (FGF) could induce mouse iPSCs to differentiate into neural crest–like cells (Otsu et al. 2012).

For the generation of salivary gland organoids, the ability of PSCs to generate new pools of salivary gland–specific cells has also been explored. ESCs were induced to differentiate into oral ectoderm, which underwent morphogenesis to form branching salivary ducts when influenced by FGF7 and FGF10 signaling (Tanaka et al. 2018).

Although PSCs have been widely applied for the building of organoids, specific protocols for the generation of oral organoids using different cocktails have not yet been identified. Several major concerns, such as the accuracy of choreographed steps of induction and possible tumorigenesis when transplanted, should be carefully considered before clinical application.

Scaffold

Specialized niches in vivo regulate cell fate and behavior. Extracellular matrix (ECM) is one of the main components of the stem cell niche and provides structural support and instructive signaling. To simulate the native ECM, several scaffolds, including 3D scaffolds made of natural and synthetic materials, as well as ECM scaffolds produced from decellularized matrices, have been used in organoid construction.

Natural Materials

The Matrigel, a natural ECM generated by mouse sarcoma cells, is a widely used basic organoid component to mimic the natural basement membrane. Matrigel and collagen gel have been used to construct oral organoids, mainly to provide a 3D environment for the formation of structures (Monteiro and Yelick 2017). The main advantage of these natural matrices is their combination of ECM components and growth factors, which efficiently promotes cell growth and differentiation (Rossi et al. 2018). However, their components’ complexity makes it difficult to precisely control the culture conditions and the reproducibility (Yin et al. 2016).

Synthetic Materials

As an alternative, several synthetic materials have been used in the construction of organoids since their biochemical and bioactive properties can be easily modified for different types of stem cells. For example, the highly porous structure of poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) have been used in the construction of tooth germ organoids to facilitate DE-DM interaction (Cai et al. 2017). Synthetic hydrogels of PLGA and hyaluronic acid (HA) used for SG organoids can promote the generation of interconnected and branched salivary gland structures (Ozdemir et al. 2016). Nevertheless, the modification of biologically active factors on the scaffolds is required to meet organoid growth needs.

ECM Scaffolds Derived from Decellularized Tissues

With the advancements in the tissue decellularization techniques, acellular tissue-derived scaffolds are proposed as an alternative to other scaffolds. Traphagen et al. (2012) have described 3 methods to effectively decellularize and demineralize porcine molar tooth buds while preserving natural ECM protein gradients. The addition of DE cells and DPSCs to acellular scaffolds resulted in the formation of enamel and dentine after transplantation (Zhang et al. 2017). Besides, the decellularized submandibular gland (SMG) could support cellular adhesion and form gland-like tissues when reseeded with allogeneic primary SMG cells (Gao et al. 2014).

In general, different materials offer unique advantages in the construction of oral organoids. Until now, no scaffold could accurately provide physiological spatial-temporal cues to replicate the process of oral organogenesis. Additional developments should be made in future studies to incorporate essential signals from native ECMs into scaffolds, optimize the decellularization process of oral organs, and explore the effective method of recellularization.

Construction Strategies for Different Oral Organoids

Oral organoid construction strategies generally depend on the interaction between seed cells and niche, which creates a dynamic environment that guides the self-renewal, differentiation, and assembly of seed cells in organoids (Clevers 2016). Here, we will describe the main strategies for generating different oral organoids.

Tooth Germ Organoids

The key point in constructing tooth germ organoids is to replicate the spatial-temporal interaction between dental epithelium and mesenchyme (Fig. 1). In a pioneer study, the combination of dissociated DE and DM cells from mandibular tooth germs at the cap stage in ED14.5 mice with a collagen gel droplet formed a tooth germ–like structure in conventional tooth germ culture medium (Nakao et al. 2007). The reconstituted tooth germ generated a complete bioengineered tooth in the tooth cavity after transplantation. This study provides the first evidence of a successful reconstitution of an entire tooth germ organoid. Besides embryonic tooth germ, autologous postnatal tooth germ has been used as a source of DE and DM cells. The tooth germ organoid successfully developed and erupted into the oral cavity in a canine model (Ono et al. 2017). The tooth germ organoids of pigs have also been created through the same method (Ono et al. 2017; Wang et al. 2018). PSC-derived DE (Cai et al. 2013) or DM (Kim et al. 2019) cells combined with dental mesenchyme or dental epithelium also generated tooth germ organoids. After transplantation in vivo, the organoids formed a tooth-like structure with physical properties such as elastic modulus and hardness similar to the human tooth.

Moreover, scaffold-free construction methods have been established. A high density of DPSCs was cultured in a low attachment plate to imitate the process of mesenchymal condensation, which leads to intercellular connections and a dense aggregate. The condensates of DPSCs and cells of epithelial origin were then cocultured in a composite medium appropriate for both cell types. After 4 wk of 3D culture, tooth germ organoids with an epithelial sheath structure around mesenchymal cells are formed (Rosowski et al. 2019). Even if there are no in vivo experiments to verify whether this tooth germ–like organoid could eventually develop into teeth, it still provides a valuable model to study underlying development mechanisms in vitro.

The difficulty of the whole-tooth organoid construction lies in the complexity of the tooth structure. The engineered tooth not only should achieve the hardness and elastic modulus of the hard tissue but also needs the blood supply and nerve regeneration in the soft tissue. To facilitate the vascularization of organoids, cells with angiogenic ability can be added to the cell mixture. Combination of human umbilical vein endothelial cells (HUVECs) with DE and DM cells in GelMA has been applied to develop vascularized tooth germ organoids (Smith et al. 2017). The implanted tooth germ organoids exhibited functional vascularization with circulating host red blood cells. Notably, the tooth germ organoid formed mineralized tissues that largely adopted the size and shape of the original constructs and expressed tooth markers. However, no distinct enamel or dentin layers were observed.

In general, these studies suggest a possibility to generate teeth from the construction of tooth germ organoids. Different construction strategies of tooth germ organoids have specific strengths and limitations (Table 1). Tooth germ organoids derived from embryonic dental cells could generate the entire structure of tooth, including hard tissue, pulp tissue, blood vessels, and nerve fibers. But this strategy is limited by the lack of source of embryonic tooth cells. The application of PSCs and ASCs might solve the source of cells in organoid construction. Finding an effective way of induction is a prerequisite for the clinical application of iPSCs in tooth germ organoids. Meanwhile, a suitable artificial scaffold mimicking the components of ECM is equally important.

Table 1.

Summary of Various Construction Strategies of Tooth Germ Organoid and Their Strengths and Limitations.

Cell Type	Scaffold	Culture System	Outcome	Strengths	Limitations	Reference
Embryonic epithelial and mesenchymal cells	Collagen gel	Culture on cell culture insert containing DMEM supplemented with 10% FCS, 100 µg/mL ascorbic acid, 2 mM L-glutamine	Form a correct tooth structure comprising enamel, dentin, root analog, dental pulp, blood vessels, and bone	Cells derived from tooth germ have the ability of self-sorting and have definite odontogenic differentiation ability; collagen is the main component of natural ECM	Lack of embryonic tooth cells and ethics issue; the collagen cannot provide appropriate stiffness to form teeth of predetermined size and shape	Nakao et al. 2007
Postnatal dental epithelial tissue and mesenchymal cells	Collagen gel	Culture on a cell culture insert in basal medium consisting of α-MEM, 10% fetal bovine serum, 100 µM L-ascorbic acid 2-phosphate	Successfully erupt into oral cavity with the same components as the natural tooth	Generate large size of tooth and realize the autologous transplantation	Lack of appropriate cell sources	Ono et al. 2017
iPSCs	Not mentioned	Coculture of dental epithelial and mesenchymal cells induced by iPSCs	Form tooth-like structure with similar elastic modulus and hardness to natural tooth	Excellent multipotent differentiation and self-renewal capabilities; free of immune rejection	The effective way of induction is coculture with tooth germ tissue	Kim et al. 2019
DPSCs and keratinocytes from gingiva	No scaffold	A single-cell suspension of epithelial cells in a ratio of 1:4 related to the initial cell number used for mesenchymal condensation is cultured under nonadherent conditions in a composite medium appropriate for both cell types	Form a tooth germ organoid the size of human tooth germ, mimicking the reciprocal tissue interactions of human tooth development	Cells are easily obtained from wisdom teeth and gingiva	Without in vivo experiment, the result of regeneration after transplantation is uncertain	Rosowski et al. 2019
Porcine dental epithelial cells, human dental pulp stem cells, and HUVECs	dTBs	Recell dTBs with cell mixture and culture in a bioreactor for 1 wk in 1:1:1 (DM/DE/HUVEC) media with osteogenic supplements	Successful formation of tooth with mature enamel, pulp, dentin, periodontal ligament, and tooth roots, which adopted the size and shape of dTBs	HUVECs support the formation of neovasculature; dTBs provide a natural extracellular matrix environment to full-sized organ regeneration	There are problems of biocompatibility and immune response in heterogeneous ECM; HUVECs can only be an allogeneic source	Zhang et al. 2017
Porcine dental epithelial and mesenchymal cells and HUVECs	GelMA	GelMA containing either pDE- HUVECs or pDM-HUVECs are cultured in osteogenic media	Form robust mineralized tissues that largely adopted the size and shape of the original construct	Tooth organoid of specified size and shape can be built	No distinct enamel or dentin layers were observed	Smith et al. 2017
Porcine dental epithelial cells and human dental pulp stem cells	Electrospun PLGA/PCL/nHA fibrous scaffolds	A mixture of pDE and hDM cells (1:1) is seeded to each scaffold and cultured in low-adhesive plates containing 1:1 (DE/DM) media with osteogenic supplements	Establish tooth germ organoid with appropriate DE-DM cell interaction	Exhibit sufficient porosity to support dental cell in growth	The residue of the material may lead to tooth structural disorder	Cai et al. 2017

α-MEM, minimum essential medium alpha; DE, dental epithelial; DM, dental mesenchymal; DMEM, Dulbecco’s modified Eagle’s medium; DPSC, dental pulp stem cells; dTB, decellularized porcine tooth bud; ECM, extracellular matrix; FBS, fetal bovine serum; hDM, human tooth pulp-derived dental mesenchymal; HUVEC, human umbilical vein endothelial cell; iPSC, induced pluripotent stem cell; nHA, nano-hydroxyapatite; PCL, polycaprolactone; pDE, pig dental epithelial; pDM, pig dental mesenchymal; PLGA, poly(lactic-co-glycolic acid).

Salivary Gland Organoids

The key to constructing salivary gland (SG) organoids lies in the recovery of salivary gland duct structure and secretory function. Two main strategies have been delineated in research on the construction of salivary gland organoids.

PSC-derived SG organoids

There has been an approach to generate embryonic SG organoids. Tanaka and colleagues (2018) used several cytokines and small molecules, including BMP4, SB-431542 (inhibitor of transforming growth factor β [TGF-β]), LDN-193189 (inhibitor of BMP), and FGF2, to induce the cells at the outer layer of ESC aggregates to differentiate into oral ectoderm. Subsequently, the aggregates were infected with recombinant adenovirus encoding Sox9 and Foxc1. The infected outer layers were isolated and cultured with FGF7 and FGF10 to promote salivary gland morphogenesis from oral ectoderm (Fig. 2A). The SG organoid formed was similar to the embryonic submandibular gland during E15 to E18 in terms of its morphological, molecular, and functional properties (Tanaka et al. 2018). Following orthotopic transplantation into mice whose SGs had been removed, the induced SG rudiment exhibited mature SG characteristics, including saliva secretion.

ASC-derived SG organoids

Recent studies have found that stem/progenitor cells isolated from the salivary gland can be expanded and differentiated to generate organoids capable of restoring gland function in vivo (Fig. 2A) (Farahat et al. 2019; Sui et al. 2020). These processes are controlled by the treatment of numerous growth factors, including EGF, FGFs, and TGF-β (Farahat et al. 2019; Sui et al. 2020). The artificial niche promoted the development of salivary gland organoids, as indicated by the expression of differentiation markers, structure formation, and response to neurotransmitters in vitro. However, the secretory function is decreased due to the absence of continuous stimulation factors (Sui et al. 2020). Another approach with fewer exogenous niche factors under 3D culture conditions has also been explored (Shin et al. 2018). This niche-independent method is a 2-step process: 1) culture of single clonal SG stem cell aggregation in DMEM supplemented with 10% FBS and 2) differentiation in serum-free hepato-STIM medium supplemented with EGF. The whole process takes place in 3D microwells. Microwells are fabricated by photopatterning poly hydrogel in the presence of an electrospun polycaprolactone nanofibrous scaffold (Fig. 2B). The generated SG organoids exhibited more prominent acinar and ductal markers than those from traditional 3D cultures. Moreover, 3D bioprinting can also be used in the construction of ASC-derived SG organoids (Fig. 2C) (Adine et al. 2018). KIT⁺ DPSCs tagged with magnetic nanoparticles were spatially arranged with magnet dots to generate 3D structures. Next, the cells were induced with FGF10 to differentiate into SG epithelial cells by recapitulating SG epithelial morphogenesis and neurogenesis. The strategy, based on magnetic 3D bioprinting incorporation, has successfully developed SG organoids with neuronal compartments, secretory function, and epithelial polarity but limited vascularization.

Although functional salivary gland organs have been constructed, several problems still need to be solved before the application of regenerative medicine (Table 2). It is necessary to determine the suitable source of stem cells and to identify the key signal molecules in the differentiation of stem cells into SG epithelial cells. The strategy to construct a vascularized salivary gland organoid has not been established yet. Further studies should focus on the construction of an SG organoid that can survive and function in the host for a long time.

Table 2.

Summary of Construction Strategies of Salivary Gland Organoid and Their Strengths and Limitations.

Cell Type	Scaffold	Culture System	Outcome	Strengths	Limitations	Reference
SIMS cell line	Matrigel	Cell/collagen/Matrigel mix is cultured in differentiation medium (SIMS complete medium with supplements such as EGF and FGF2)	Salivary gland organoid formation after single-cell culture is apparent around day 5 of culture	SIMS cell line can differentiate into unique populations of acinar, myoepithelial, and duct cells; induce stem/progenitor cells differentiation by causing cell polarization	The complex components of Matrigel make controlling culture conditions difficult and may reduce reproducibility	Athwal et al. 2019
Embryonic SMG mesenchymal and epithelial cells	Matrigel/Matrigel modified by fibronectin	Cell pellets are seeded in a drop of Matrigel/Matrigel with a supplement of FN and FGF7	Build an SMG organoid model that accounts for the dynamic interactions between the dissociated epithelial and mesenchymal cells	Cells derived from embryonic SMG have strong self-organization ability; fibronectin can mediate cellular adhesion, migration, growth, and differentiation	The cells do not differentiate to full maturity; lack of cell source	Hosseini et al. 2019;Farahat et al. 2019
Rat primary SMG cells	Decellularized rat submandibular glands	Scaffold with cells reseeded is put into a culture vessel of a rotary cell culture system with mammary epithelial growth medium	Form duct-like structures with a potential secretory function	The scaffold can provide the native spatial structure but also guides proper cellular interactions by releasing molecular cues	Mesenchymal cells are rarely observed even with a mix of gland cells seeded as described	Gao et al. 2014
Human submandibular gland stem/progenitor cells and mouse embryonic salivary gland mesenchyme	Matrigel	The cell suspension is injected into a Matrigel drop and cultured in medium with FDF10 added	Generate mature salivary glands, including a natural morphology and saliva secretion	Homologous and low risk	Growth factors need to be provided continuously	Sui et al. 2020
DPSCs	No scaffold	The cells are bioprinted into spheres and cultured in epithelial differentiation medium supplemented with FGF10	Generate salivary gland tissue with secretory function and neuronal integration	DPSCs are easy to obtain; using magnetic 3-dimensional bioprinting allows cellular spatial arrangement and spheroid formation within less than 24 h	Vascularization is limited in the organoids	Adine et al. 2018
Human single clonal salivary gland stem cells	No scaffold	Cells are preconditioned to aggregate to form 3-dimensional spheroids in microwells, after which they are cultured in serum-free hepato-STIM medium supplemented with EGF	Form more functional salivary gland organoids with higher α-amylase secretion and intracellular calcium levels	Nanofibrous microwell culture system allows no exogenous niche factors added	Not mentioned	Shin et al. 2018
Embryonic stem cells	Collagen gel	Cell aggregations are induced into oral ectoderm by supplements such as BMP4 and FGF2; then, the out layers infected with adenovirus encoding Sox9 and Foxc1 are cultured with FGF7 and FGF10	The salivary gland organoids secrete saliva after orthotopic transplantation in mice	It provides a strategy to induce salivary gland organoid organogenesis and maturation without coculture with mesenchyme	Excessive growth factors and accurate regulation are needed	Tanaka et al. 2018

DPSC, dental pulp stem cell; EGF, epidermal growth factor; FGF, fibroblast growth factor; FN, fibronectin; SMG, submandibular gland.

Lingual Epithelium Organoids

A lingual epithelium organoid culture system has been established (Fig. 3A) (Hisha et al. 2013). The Bmi1⁺ LESCs released from the lingual fragments are cultured in Matrigel supplemented with cytokines, including (EGF), noggin, and R-spondin 1. Under these conditions, 3 different types of organoids are generated: round-shaped organoids with concentric cell arrangements and rugged- and round-shaped organoids with a reticulated cell arrangement. Among them, the round-shaped organoids with concentric cell arrangements have a multilayer keratinized epithelium and a stratum corneum, which are characteristic of filiform papillae. Immature organoids harvested after 3-d culture in recipient mouse tongues formed a concentrically structured stratum corneum, indicating possible application in regenerative medicine.

The organoid culture system allows it to generate a stratified keratinized epithelial cell layer from cells isolated from lingual fragments. However, this system’s limitation is the heterogeneity of organoids generated under the same cultural conditions (Hisha et al. 2016). After transplantation, the grafted organoids expanded in the muscle layer rather than the epithelial layer of the recipients’ tongues. Therefore, the technique to ensure the development of lingual epithelium organoid in the subepithelium area is needed before the application in lingual regeneration.

Taste Bud Organoids

Taste buds are independent organs on the tongue, and the loss of taste affects quality of life. Taste bud organoids can be generated from Lgr5⁺ or Lgr6⁺ taste bud stem cells or circumvallate papilla tissues (Fig. 3B). Lgr5⁺ or Lgr6⁺ cells are sorted from taste tissues, and culture for taste bud organoids is based on DMEM/F12 supplemented with R-spondin 1, Noggin, Jagged 1, Y27632, N-acetylcysteine, EGF, N2, and B27. Sorted cells in the medium are mixed with an equal volume of chilled Matrigel and then seeded on the organoid culture plates (Ren et al. 2014). In addition, with the supplementation of Wnt, EGF, R-spondin 1, and Noggin, taste bud organoids can be constructed from circumvallate papilla tissues (Aihara et al. 2015). These taste bud organoids develop into phenotypic characteristics similar to natural tissues, including multiple layers of epithelial cells containing stem/progenitor cells in the outer layer and taste cells in the inner layer.

Potential Applications of Oral Organoids

Oral organoids have immediate potential applications. Oral organoids provide great models for in vitro and in vivo experiments in addition to the development of personalized medicine for the treatment of diseases. In regards to regenerative medicine, oral organoids are a promising source of transplantable tissues and functional cell types for therapy.

Oral Organoids as Models of Development

The formation of PSC-derived organoids mirrors natural organogenesis to some extent and provides an in-depth understanding of the process in addition to exhibiting greater experimental accessibility than animal models. For example, tooth germ organoids display odontogenic markers and are also capable of epithelial invagination into the condensed mesenchyme, mimicking reciprocal tissue interactions of human tooth development. Comprehensive transcriptome analysis has also indicated the activation of signaling pathways involved in human tooth organogenesis, such as Notch and TGF-β signaling (Smith et al. 2017; Rosowski et al. 2019). In the construction of SG organoids, FGF2-dependent mesenchyme and laminin 111 were identified to be the essential niche factors in promoting SG cells maturation, and transcription factors (Sox9 and Foxc1) are responsible for the differentiation of oral ectoderm into the salivary gland rudiment (Tanaka et al. 2018; Hosseini et al. 2019).

Oral Organoids for Disease Modeling

To study diseases, oral organoids can be directly derived from patients’ cells (Schutgens and Clevers 2020). The application of organoids in tumor research shows particularly outstanding advantages in the precision treatment of oral cancers. Unlike monolayer cell lines, organoids are genetically stable during long-term culture and capture the original tumor’s partial heterogeneity. Tongue cancer is one of the major malignant cancers in the maxillofacial region. Tongue squamous cell carcinoma (TSCC) organoids have been created by seeding TSCC cell line CAL27 into the decellularized tongue extracellular matrix (TEM) (Zhao et al. 2017). Briefly, TEM was cultured with tongue CAL27 for 14 d. Next, cancer-associated fibroblasts (CAFs) mixed with CAL27 were microinjected into the muscle layer of TEM and cultured for 28 d. The results show that the TEM-reconstructed TSCC organoid presented better histopathological characteristics of TSCC than the Matrigel culture system (Fig. 3C). The ability of TSCC organoids to model tumor pathologies will provide great opportunities to study the feasibility of drug testing and screening applications (Zhao et al. 2017). Paired normal and cancer organoids can be established from the same patient samples, thereby providing an opportunity for personalized medicine. Moreover, taste bud organoids have been used to explore the effects of radiation on taste buds, suggesting SIRT1 inhibitors can promote Lgr5⁺ taste bud stem cell survival and mitigate radiation-induced oral mucositis (Guo et al. 2019). SG organoids have also made progress in mimicking radiation damage in studying the mechanism of stem cell response to radiation (Serrano Martinez et al. 2020).

Regenerative Medicine

Donated tissues and organs for transplantation are always scarce. As organoids are initiated from minuscule amounts of donor cells, expanded and differentiated in vitro, they can avoid immune rejection and ethical issues and become a suitable source for transplantation in the future. Orthotopically engrafted SG organoids exhibit rebuilding of the nervous system, resulting in full saliva secretion, suggesting the great potential for repair and regeneration of SGs (Tanaka et al. 2018).

Although tooth germ organoids have not yet developed into perfect teeth, they provide a new method for creating intact teeth. Creating a bioengineered tooth germ organoid that may be cultured in vitro and transplanted into the jawbone to form a fully functional tooth will be useful for clinical therapy. Tongue tissue defects caused by cancer and trauma cause great difficulties in chewing and swallowing, affecting quality of life. The lingual epithelium organoid culture system has provided a convenient and straightforward method for understanding the mechanism of maintenance and hyperplasia of lingual epithelial tissue, which is important in studying tongue cancer and regeneration.

Future Directions

Although oral organoid-related technologies have dramatically developed in the past decades, they have yet to become perfect models of native organs. It is not clear to what extent they can functionally replace the original organs after being transplanted into the human body. Different oral organoids have very distinct structures and functions, so there is no general construction strategy for all organoids. Therefore, a better understanding of the molecular mechanisms of organ development and tumorigenesis could then be applied to drive the development of more mature organs and disease models.

The main challenge that needs to be addressed is the source of cells. Although the cells derived from embryonic tissues achieved great development in experiments, they are limited in application due to the lack of cell source and ethical issues. Although autologous iPSCs are used as the potential cell source to construct oral organoids in the future, the efficiency, stability, and reproductivity of the organoid culture system to generate specific cell types still need to be improved (Huch et al. 2017).

Besides epithelial and mesenchyme components, a fully developed organoid also needs a vascular system and neuralization. Although HUVECs have been used to vascularize the dental pulp, the cell source is allogeneic. Vessel organoids constructed by autologous iPSCs can try to be used in the construction of oral organoids. For tooth and tongue with complex structures, bioengineering tools (such as biomimetic scaffolds and 3D bioprinting) can be used to achieve an orderly combination of different components (Gjorevski et al. 2016; Park et al. 2019). Stem cells can also be engineered by genome editing to elucidate signal pathways of the organogenesis and disease development (Miura and Suzuki 2017; Nie and Hashino 2017). Using these methodologies, cells’ fate can be regulated by manipulating exogenous niche components and reprograming the internal decision-making structure (Fig. 4).

Conclusion

Oral organoids represent an important bridge between 2D cell line cultures and in vivo animal models: they can recapitulate the 3D structure of the targeted organ and at the same time are relatively accessible and stable. Currently, several different approaches have successfully constructed different oral organoids, which have already been used for experiments in oral tissue regeneration and oral disease treatment. Oral organoids may become an alternative for the transplantation of oral organs and help us understand the underlying mechanism of human oral development and oral diseases.

Author Contributions

X. Gao, contributed to conception, design, and data analysis, drafted the manuscript; Y. Wu, contributed to data analysis, drafted the manuscript; L. Liao, W. Tian, contributed to conception, design, and data analysis, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material

sj-pdf-1-jdr-10.1177_0022034520983808 – Supplemental material for Oral Organoids: Progress and Challenges

Supplemental material, sj-pdf-1-jdr-10.1177_0022034520983808 for Oral Organoids: Progress and Challenges by X. Gao, Y. Wu, L. Liao and W. Tian in Journal of Dental Research

Footnotes

A supplemental appendix to this article is available online.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Key Research and Development Program of China (2017YFA0104800), the Nature Science Foundation of China (82071092, 31601113), the Fundamental Research Funds for the Central Universities (YJ201878), and Key Project of Sichuan province (2019YFS0311, 2019YFS0515).

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