Journal of Clinical Pediatric Dentistry,2023,47(6):74-85 DOI:10.22514/jocpd.2023.081
Original Research

Stemness maintenance of stem cells derived from human exfoliated deciduous teeth (SHED) in 3D spheroid formation through the TGF-β/Smad signaling pathway

Hongwen Li1,2,†, Jing Jiang1,†, Haiying Kong1, Wenbo Wu1,2, Xiaomin Shao3, Shuqi Qiu1, Xianhai Zeng1, Qinghong Zhong1, Xinhui Yao1, Xiantao Zeng1, Lingshan Gou4, Jian Xu1,2,*,

1Longgang E.N.T. Hospital & Shenzhen Key Laboratory of E.N.T., Institute of E.N.T., 518172 Shenzhen, Guangdong, China

2Shenzhen Longgang Institute of Stomatology, 518172 Shenzhen, Guangdong, China

3Longgang District People’s Hospital of Shenzhen, 518116 Shenzhen, Guangdong, China

4Xuzhou Maternity and Child Health Care Hospital, 221009 Xuzhou, Jiangsu, China

*Corresponding Author(s):xj-sz@hotmail.com (Jian Xu)

† These authors contributed equally.

History Submitted: 26 December 2022 | Accepted: 12 May 2023 | Published: 03 November 2023
Copyright:  ©2023  The Author(s). Published by MRE Press.
This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

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Abstract

Mesenchymal stem cells (MSCs) have shown great potential as important therapeutic tools for dental pulp tissue engineering, with the maintenance and enhancement of their stemness being crucial for successful therapeutic application in vivo and three-dimensional (3D) spheroid formation considered a reliable technique for enhancing their pluripotency. Human exfoliated deciduous tooth stem cells (SHED) were cultured in a low attachment plate to form aggregates for five days. Then, the resulting spheroids were analyzed for pluripotent marker expression, paracrine secretory function, proliferation, signaling pathways involved, and distribution of key proteins within the spheroids. The results indicated that 3D spheroid formation significantly increased the activation of the transforming growth factor beta (TGF-β)/Smad signaling pathway and upregulated the secretion and mRNA expression levels of TGF-β, which in turn enhanced the expression of pluripotency markers in SHED spheroids. The activation of the TGF-β/Smad signaling pathway through 3D spheroid formation was found to preserve the stemness properties of SHED. Thus, understanding the mechanisms behind pluripotency maintenance of SHED culture through 3D spheroid formation could have implications for the therapeutic application of MSCs in regenerative medicine and tissue engineering.

Keywords:3D spheroid culture;Stemness maintenance;Stem cells derived from human exfoliated deciduous teeth (SHED);TGF-β/Smad signaling
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Cite this article

Hongwen Li, Jing Jiang, Haiying Kong, Wenbo Wu, Xiaomin Shao, Shuqi Qiu, et al.Stemness maintenance of stem cells derived from human exfoliated deciduous teeth (SHED) in 3D spheroid formation through the TGF-β/Smad signaling pathway.Journal of Clinical Pediatric Dentistry,2023,47(6):74-85 DOI:10.22514/jocpd.2023.081

1. Introduction

Dental pulp, a highly organized soft connective tissue located within the mineralized structure of the tooth, plays a pivotal role in maintaining tooth homeostasis and serves as a vital organ for the tooth’s viability [1]. Pulpitis is common in dental pulp, often caused by deep decay, dental trauma or advanced periodontal disease, and is traditionally treated by root canal therapy, which involves removing the affected pulp [2]. Recent studies have shown that mesenchymal stem cell (MSC)-based tissue regeneration is promising for engineering dental pulp tissue and restoring tooth viability [3, 4]. Further, in both nude mice ectopic and dogs or minipigs orthotopic regeneration models, the transplantation of stem cells from the apical papilla (SCAPs) or dental pulp stem cells (DPSCs) resulted in the regeneration of blood vessels, remodeling and dentin-pulp-like tissues [5, 6, 7].

In recent years, stem cells derived from human exfoliated deciduous teeth (SHED) have emerged as a promising candidate among MSCs for treating diseased dental organs [8, 9]. Compared to other MSC types, SHED has numerous advantages, including strong proliferative potential, higher cellular differentiation, minimal ethical concerns and less invasive procurement [10, 11]. The origin of SHED from the dental pulp of exfoliated deciduous teeth, which is in the developmental stage, is one of the reasons for its distinct characteristics. As exfoliated deciduous teeth are shed at a relatively early stage of tooth development, their pulp tissues may harbor stem cells possessing greater pluripotency, self-renewal ability and more effective differentiation than those derived from mature dental tissues or other adult MSCs. In addition, SHED possesses unique advantages, such as high levels of specific pluripotency marker expression and the ability to secrete a broad array of growth factors and cytokines [12, 13, 14, 15]. Thus, SHED has been successfully applied therapeutically for repairing/regenerating both the local intraoral pulp-dentin complex and extraoral tissues. In nude mice models, the transplantation of tooth slices or hydroxyapatite/tricalcium phosphate (HA/TCP) containing SHED resulted in the formation of dentin-pulp-like tissues in vivo. In addition, the implantation of SHED with either recombinant human Collagen type I or PuraMatrix (peptide hydrogel) into the premolar roots of immunodeficient mice promoted the regeneration of pulp-like tissues with new tubular dentin [3, 16, 17, 18, 19]. Nevertheless, since many therapeutic applications have been tested in animal models, there is an urgent need for conducting extensive (pre-)clinical trials [1, 20].

The mesenchymal cell population in the craniofacial region during embryogenesis mainly originates from the neural crest of the ectoderm [21, 22, 23]. MSCs obtained from dental pulp tissues exhibit specific markers for embryonic stem cells, such as transcription factors SRY-box transcription factor 2 (SOX2), octamer-binding transcription factor 4 (OCT4) and (NANOG), and cell surface antigen Stage-specific embryonic antigen-4 (SSEA-4) [24, 25]. In addition, expressing pluripotency markers similar to embryonic stem cells (ESCs) could potentially enhance the effectiveness of stem cell therapy-based treatments [26]. However, the successful application of dental MSCs in regenerative medicine and tissue engineering heavily depends on the ability to regulate stemness properties and maintain pluripotency throughout multiple subcultures from the initial cells [27, 28]. Although the pluripotency of MSCs is maintained in the in vivo microenvironment through various mechanisms and factors such as cell-cell contacts, soluble growth factors and interactions with the extracellular matrix (ECM), these primitive stemness properties can gradually diminish over time during conventional monolayer tissue culture in vitro [29].

Cells in suspension can form three-dimensional (3D) spheroids, allowing for significant multiplanar interactions with the cell matrix, which create niches and microenvironments that influence cellular processes such as cell function and fate [30, 31]. MSCs cultured in spheroids exhibit a distinctive morphology, characterized by a spherical shape on the inside and an elongated shape on the outside. In contrast to conventional 2D monolayer cultures, they display reduced levels of extracellular matrix (ECM) and cytoskeletal molecules and a 75% reduction in individual cell volume [32, 33]. McBeath et al. [34] reported that MSCs with a small, rounded morphology were more likely to differentiate into an adipogenic lineage, thereby highlighting the importance of cellular morphology in determining cellular fates and phenotypes [35]. The formation of 3D spheroids is associated with several advantages, including the ability to study authentic in vivo cellular function and differentiation through in vitro cell culture [36, 37]. In addition, 3D cell cultures offer a rapid way to conduct experimental manipulations using molecular biology techniques and allow for superior imaging through microscopy compared to animal models [38]. Recent evidence suggests that MSCs cultured in 3D spheroids may enhance angiogenic, anti-inflammatory, multipotency and tissue regenerative effects both in vivo and in vitro as well as increasing the secretion of various factors, including cytokines, vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), stanniocalcin-1 (STC-1) and tumor necrosis factor-α stimulated gene/protein 6 (TSG-6) [12, 39, 40]. Microarray analysis has revealed a notable shift in gene expression profiles during 3D spheroid formation, marked by the upregulation of genes related to inflammation, angiogenesis, redox signaling, stress response and hypoxia [40, 41]. Moreover, MSCs cultured as 3D spheroids have shown enhanced therapeutic effects in diverse disease models, such as ischemic injury and wound healing, owing to the above-mentioned stem cell biological properties [42, 43, 44]. Thus, the primary objective of tissue engineering is to imitate the complex geometric and architectural characteristics of the body, including critical cell-to-cell interactions. In this regard, various techniques have been devised for regenerative medicine, among which the most widely used approach involves incorporating biological factors such as cells, proteins and genes into porous, degradable scaffold materials [45, 46, 47]. Despite the advantages of using scaffold materials, it is important to note that scaffold environments may not completely replicate the natural cell-to-cell and cell-to-ECM attachments observed in vivo tissues. Furthermore, there have been reports of infection and inflammation resulting from scaffold materials during in vivo degradation [48]. In 3D spheroid culture, the cells and their extracellular matrix (ECM) can self-assemble into 3D aggregations without a scaffold. Scaffold-free 3D cell culture techniques have gained substantial interest in stem cell research because they can simulate the in vivo microenvironment, improve the potency of MSCs, avoid the risk of infection and/or inflammation, and promote increased cell-to-cell interactions [49, 50]. Therefore, considering the current trends in cell biology and tissue engineering, investigating the stemness of SHED cells in 3D spheroids is a timely and relevant research topic.

The transforming growth factor β (TGF-β) superfamily plays a crucial role in regulating numerous cellular processes in MSCs. The signaling process begins with TGF-β factors binding to a cell surface receptor complex consisting of two pairs of receptor serine/threonine kinases, specifically types I and II. The type II receptor then phosphorylates the type I receptor, which induces the signal by phosphorylating receptor-activated Smad (R-Smads) proteins, including Smad2/3 (TGF-β-responsive Smads) and Smad1/5/8 (bone morphogenetic protein (BMP)-responsive Smads). R-Smad proteins are primarily present in the cytoplasm [51]. Once activated, R-Smad proteins form a trimeric complex with Smad4 in the cytoplasm and nucleus, which then enters the nucleus to regulate gene expression. Studies have shown that TGF-β-responsive Smads can bind to the NANOG proximal promoter, and recent research indicates that TGF-β/Smad signaling enhances the activity of the NANOG promoter to sustain the stemness properties of human embryonic stem cells [52]. In addition to human ESCs, studies have reported that TGF-β/Smad signaling can regulate the expression of NANOG in mouse pluripotent stem cells derived from the epiblast layer of pre-gastrula stage mouse embryos (EpiSCs) [53].

Furthermore, MSCs cultured in 3D spheroid formation have been shown to preserve the epigenetic status of pluripotent genes [54]. Additionally, they synthesize significantly higher levels of TGF-β or BMP2 compared to monolayer cultures, leading to the activation of the TGF-β/Smad signaling pathway [55, 56]. Herein, we hypothesize that the TGF-β/Smad signaling pathway plays a crucial role in altering pluripotent gene expressions in MSCs cultured in 3D spheroids. Therefore, we investigated the impact of 3D spheroid formation on pluripotency and analyzed the function of the TGF-β/Smad signaling pathway in modifying the epigenetic status of pluripotent genes. By understanding the mechanisms behind the maintenance of pluripotency in SHED cultured in 3D spheroids, we hope to make significant contributions to the therapeutic applications of MSCs in regenerative medicine and tissue engineering.

2. Materials and methods

2.1 Cell isolation and culture

SHEDs were isolated from clinically extracted deciduous teeth of three healthy patients aged 6–9 years old. The isolation procedure was performed using a standard enzymatic digestion protocol, and the isolated SHEDs were characterized according to the method described by Miura et al. [57]. SHED was cultured in Eagle’s minimum essential medium alpha modification (α-MEM; GIBCO, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin solution (Invitrogen, Carlsbad, CA, USA) at 37 °C in 5% carbon dioxide (CO2). The cells were sub-cultured at a ratio of 1:6 when they reached 80% confluence, and the culture medium was changed every 2–3 days. SHED of passages 4–5 were used for all experiments.

2.2 3D spheroid formation

SHED were cultured in a low attachment 6-well plate (Corning, Corning, NY, USA) at a density of 100,000 cells/well. For comparison, cells were also cultured in standard plastic dishes (Corning, Corning, NY, USA) as a control. The SHED spheroid colonies were harvested for further assays at 1, 3 and 5 days (n = 3). The number and size of spheroids were evaluated under an inverted microscope with 10× magnification at two-time points: 3 and 5 days. Three independent experiments were conducted, and for each observation, six randomly selected fields of a 6-well plate were used. The number of spheroids was counted, and the size of the spheroids was measured in each field. To assess cell proliferation, a cholecystokinin octapeptide (CCK-8)-based assay was performed (Dojindo Molecular Technologies, Inc. Kumamoto, Japan) following the manufacturer’s protocol.

2.3 Enzyme-linked immunosorbent assay (ELISA)

The supernatant was collected from SHED cultured in spheroids or monolayers for 1, 3 and 5 days and stored at −80 °C for later experiments. To assess the levels of secreted TGF-β, the supernatants were centrifuged at 15,000 × g for 10 minutes at 4 °C, and a Human TGF-β ELISA kit (R&D Systems, Minneapolis, MN, USA) was used according to the manufacturer’s instructions.

2.4 Real-time quantitative PCR (qRT-PCR) analysis

To analyze the gene expression, SHEDs were cultured in spheroids or monolayers for 1, 3 and 5 days before being harvested. To investigate the role of TGF-β/Smad signaling pathways in 3D spheroid formation, the specific inhibitor of Smad3 (SIS3, 2 µM) was added to the low attachment culture plate according to the manufacturer’s instructions. Total RNA was extracted from spheroids and monolayers using TRIzol® reagent (Invitrogen, Burlington, ON, Canada). The expression levels of pluripotency markers, including OCT4, NANOG, SOX2 and TGF-β, were analyzed using real-time quantitative polymerase chain reaction (PCR). cDNA was synthesized from RNA using the PrimScriptTM II 1st strand cDNA Synthesis Kit (TaKaRa, Kyoto, Japan). Real-time PCR was performed to amplify the cDNA of target genes using the SYBR Premix Ex Taq II Kit (TaKaRa, Dalian, China) and primers listed in Table 1.

Table 1.Primer sequences used for the real-time quantitative PCR.
Target genePrimer sequences
GAPDH
Forward5′-GGCATGGACTGTGGTCATGAG-3′
Reverse5′-TGCACCACCAACTGCTTAGC-3′
OCT4
Forward5′-AGCAAAACCCGGAGGAGT-3′
Reverse5′-CTATATGTGTCCGGCTACACC-3′
NANOG
Forward5′-CCTGTGATTTGTGGGCCTG-3′
Reverse5′-GACAGTCTCCGTGTGAGGCAT-3′
SOX2
Forward5′-GCCGAGTGGAAACTTTTGTCG-3′
Reverse5′-GGCAGCGTGTACTTATCCTTCT-3′
TGF-β
Forward5′-CTAATGGTGGAAACCCACAACG-3′
Reverse5′-TATCGCCAGGAATTGTTGCTG-3′
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; OCT4: octamer-binding transcription factor 4; SOX2: SRY-box transcription factor 2; TGF-β: Transforming growth factor beta.

2.5 Western blot analysis

After culturing SHED in either spheroids or monolayers, they were washed with Dulbecco’s phosphate-buffered saline (DPBS) and treated with radioimmunoprecipitation assay (RIPA) buffer (Thermo, Rockford, IL, USA) supplemented with a protease inhibitor cocktail (Thermo, Rockford, IL, USA). The supernatants were collected from the lysates after centrifugation at 12,000 × g for 15 minutes. Then, we used a PierceTM BCA Protein Assay Kit (Thermo, Janakpuri, DL, India) to determine the protein concentrations. Proteins were separated by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk (wt/vol) in 1% Tris Buffered Saline Tween-20 (TBST) solution. Antibodies against phospho-Smad3, Smad2, Smad3, Smad4 and β-actin obtained from Cell Signaling Technology were used for Western blotting. After overnight incubation with primary antibodies at 4 °C and extensive washing in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK) for 1 hour. Proteins were detected by chemiluminescence using an ECL solution (ATTO Corporation, Tokyo, Japan).

2.6 Immunofluorescence

The spheroids were washed and fixed in 4% paraformaldehyde at 4 °C overnight on the 5th day of culture. Then, they were treated with 1× Polybutylene Succinate (PBS) containing 1% Triton X-100 (Sigma-Aldrich, Rehovot, Israel) and 10% FBS at room temperature for 1 hour. Next, primary antibodies against phospho-Smad3 and Smad3 (Abcam, Cambridge, UK) were added and incubated overnight at 4 °C. After washing with 1× PBS, samples were incubated with a secondary antibody, goat anti-rabbit IgG-Alexa-488-conjugated (Invitrogen, Eugene, OR, USA), in the dark for 2 hours at room temperature. Their nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and images were captured using a Leica confocal microscopy system (version 2.61, Leica, Wetzlar, Germany).

2.7 Statistics

The analyses were performed using SPSS statistical software (version 20.0, IBM Corp., Armonk, NY, USA). The number of samples used in the experiments is indicated in each figure legend, and the data are shown as mean ± scanning electron microscopy (SEM). Statistical analyses were performed using repeated-measures one-way analysis of variance (ANOVA) with post-hoc Tukey’s test for multiple comparisons. Paired t-tests were used to compare two sets of independent sample data. A p-value of less than 0.05 was considered statistically significant.

3. Results

3.1 Spheroid formation of SHED

The SHED cells cultured in a low attachment 6-well plate began to form multicellular aggregates within 1 day, which then gradually became irregular spheroids (Fig. 1A). By day 3, various sizes of SHED spheroids were observed, and the number of spheroids increased gradually, while the number of suspended cells continuously decreased (Fig. 1B). On day 5, the SHED aggregates formed compact and round-shaped multicellular spheroids, with the number of spheroids plateauing and no obvious suspended cells in the medium (Fig. 1C). In the treatment group with SIS3, the cell morphology, number and size of spheroids were consistent with the cells in the normal medium (Fig. 1D–H).

The morphologic characteristics of SHED spheroids. (A–C): Morphology of SHED spheroids cultured in 3D spheroid formation on days 1 (A), 3 (B) and 5 (C). (D–F): Appearance of SHED spheroids cultured in 3D spheroid formation with SIS3 on days 1 (D), 3 (E) and 5 (F). The scale bar represents 100 µm. The average diameter of spheroids increased on days 3 and 5 without statistically significant differences in the two different groups (G). Although a decrease in spheroid number was observed between days 3 and 5, no significant difference was observed between the groups of 3D spheroid formation with or without SIS3 (H). 3D: three-dimensional. SIS3: specific inhibitor of Smad3.

Fig. 1.The morphologic characteristics of SHED spheroids. (A–C): Morphology of SHED spheroids cultured in 3D spheroid formation on days 1 (A), 3 (B) and 5 (C). (D–F): Appearance of SHED spheroids cultured in 3D spheroid formation with SIS3 on days 1 (D), 3 (E) and 5 (F). The scale bar represents 100 µm. The average diameter of spheroids increased on days 3 and 5 without statistically significant differences in the two different groups (G). Although a decrease in spheroid number was observed between days 3 and 5, no significant difference was observed between the groups of 3D spheroid formation with or without SIS3 (H). 3D: three-dimensional. SIS3: specific inhibitor of Smad3.

3.2 TGF-β transcript and secretion levels and proliferation properties of SHED in spheroids and monolayers

3D spheroid formation of MSCs regulates their properties of stemness and multipotency. It was found that TGF-β was involved in targeting regulatory signaling pathways that control MSCs differentiation and lineage fate determination [58]. To test the relative mRNA expressions of TGF-β in SHED spheroids at different time points, the transcript levels of TGF-β were upregulated with time during the 5 days of spheroid culture (Fig. 2A). On the other hand, the mRNA levels of TGF-β decreased significantly over the course of 5 days of monolayer culture (Fig. 2B). Based on the changes in mRNA expressions, ELISA assay demonstrated that secreted TGF-β level was markedly increased in culture medium after 1, 3 and 5 days of 3D spheroid formation (Fig. 2C). The level of secreted TGF-β in the monolayer culture medium increased markedly on day 3 but decreased on day 5 (Fig. 2D). CCK-8 showed that cells in monolayers exhibited more robust proliferative capacity; statistical differences were observed from day 1 to day 5. However, cell proliferation of SHED in spheroids was inhibited in both groups with and without SIS3 (Fig. 2E).

Relative mRNA expressions of TGF-β, soluble TGF-β level and cell proliferation of SHED in spheroids and monolayers. (A) the mRNA levels of TGF-β were markedly increased with time during the 5 days of 3D spheroid culture. (B) the mRNA levels of TGF-β were significantly decreased with time during the 5 days of monolayer culture. (C) ELISA experiments were conducted to target soluble TGF-β1 in culture medium after 1, 3 and 5 days of 3D spheroid formation (n = 9, 3 independent experiments, each with 3 technical repeats). (D) soluble TGF-β1 in the culture medium was measured via ELISA experiments on days 1, 3 and 5 of monolayer culture. (E) the proliferation of SHED was evaluated by CCK-8 assay on days 1, 3 and 5 in both monolayers and spheroids with or without SIS3 treatment (*p < 0.05). TGF-β: transforming growth factor β.

Fig. 2.Relative mRNA expressions of TGF-β, soluble TGF-β level and cell proliferation of SHED in spheroids and monolayers. (A) the mRNA levels of TGF-β were markedly increased with time during the 5 days of 3D spheroid culture. (B) the mRNA levels of TGF-β were significantly decreased with time during the 5 days of monolayer culture. (C) ELISA experiments were conducted to target soluble TGF-β1 in culture medium after 1, 3 and 5 days of 3D spheroid formation (n = 9, 3 independent experiments, each with 3 technical repeats). (D) soluble TGF-β1 in the culture medium was measured via ELISA experiments on days 1, 3 and 5 of monolayer culture. (E) the proliferation of SHED was evaluated by CCK-8 assay on days 1, 3 and 5 in both monolayers and spheroids with or without SIS3 treatment (*p < 0.05). TGF-β: transforming growth factor β.

3.3 Expression of pluripotent markers of SHED in spheroids and monolayers

To investigate the effects of 3D spheroid culture on the pluripotency of SHED, the expression levels of pluripotency genes were analyzed after 5 days of culture (Fig. 3A–C). The results showed that the expression patterns of OCT4, NANOG and SOX2 in SHED spheroids tended to increase on days 3 and 5 compared to day 1. Interestingly, the mRNA expression levels of OCT4, NANOG and SOX2 were significantly reduced in SHED spheroids treated with SIS3, indicating that the TGF-β/Smad signaling pathway mediated the maintenance of pluripotency markers in SHED spheroids. In contrast, the mRNA expression patterns of pluripotency genes in SHED cultured in monolayers for 5 days differed from those in SHED spheroids (Fig. 3D). Then, OCT4, NANOG and SOX2 mRNA levels of SHED in monolayers were examined by qRT-PCR. The results indicated that the mRNA expression levels of OCT4 did not change significantly, while NANOG mRNA levels were significantly decreased on days 3 and 5 compared to day 1. The mRNA expression levels of SOX2 were significantly reduced on day 3 but slightly upregulated on day 5 compared to day 1.

Relative mRNA expressions of pluripotency markers of OCT4, SOX2 and NANOG of SHED in spheroids and monolayers treated with or without SIS3. (A): The mRNA expression levels of OCT4 in SHED spheroids were significantly reduced when treated with SIS3 compared to the untreated group during the 5 days of 3D spheroid culture. (B): The mRNA expression levels of SOX2 in SHED spheroids were lower when treated with SIS3 compared to the untreated group during the 5 days of 3D spheroid culture. (C): The mRNA expression levels of NANOG in SHED spheroids were considerably reduced when treated with SIS3 compared to the untreated group during the 5 days of 3D spheroid culture. (D): The mRNA expression levels of OCT4, NANOG and SOX2 were measured on days 1, 3 and 5 of monolayer culture. (*p &lt; 0.05). SIS3: specific inhibitor of Smad3. OCT4: octamer-binding transcription factor 4. SOX2: SRY-box transcription factor 2.

Fig. 3.Relative mRNA expressions of pluripotency markers of OCT4, SOX2 and NANOG of SHED in spheroids and monolayers treated with or without SIS3. (A): The mRNA expression levels of OCT4 in SHED spheroids were significantly reduced when treated with SIS3 compared to the untreated group during the 5 days of 3D spheroid culture. (B): The mRNA expression levels of SOX2 in SHED spheroids were lower when treated with SIS3 compared to the untreated group during the 5 days of 3D spheroid culture. (C): The mRNA expression levels of NANOG in SHED spheroids were considerably reduced when treated with SIS3 compared to the untreated group during the 5 days of 3D spheroid culture. (D): The mRNA expression levels of OCT4, NANOG and SOX2 were measured on days 1, 3 and 5 of monolayer culture. (*p < 0.05). SIS3: specific inhibitor of Smad3. OCT4: octamer-binding transcription factor 4. SOX2: SRY-box transcription factor 2.

3.4 3D spheroid formation regulate the TGF-β/Smad signaling pathway

To investigate the potential involvement of the TGF-β/Smad signaling pathway in maintaining pluripotency of SHED in 3D spheroids, we performed Western blot analysis to assess the expression levels of Smad2, Smad3, phospho-Smad3 (P-Smad3) and Smad4. The results showed that the expression of P-Smad3 was significantly increased in SHED cultured for 5 days in 3D spheroids compared to the SIS3 treatment and 2D monolayer groups, indicating the initiation of the TGF-β/Smad signaling pathway in 3D spheroid formation. Similarly, protein expressions of Smad2 and Smad4 were significantly enhanced in the 3D spheroids group compared to the other two groups. However, the protein level of Smad3 was not changed (Fig. 4). These findings suggest that 3D spheroid formation of SHED activates the TGF-β/Smad signaling pathway, resulting in increased expression levels of P-Smad3, OCT4, NANOG and SOX2, which help to maintain the stemness properties.

Western blot analysis of the expressions of Smads in monolayers and spheroid-cultured treated with or without SIS3 for 5 days. P-Smad3: phospho-Smad3. SIS3: specific inhibitor of Smad3.

Fig. 4.Western blot analysis of the expressions of Smads in monolayers and spheroid-cultured treated with or without SIS3 for 5 days. P-Smad3: phospho-Smad3. SIS3: specific inhibitor of Smad3.

3.5 Representative immunostaining of Smads in SHED spheroids

To determine the impact of 3D spheroid formation on Smads, immunofluorescence assays were performed on spheroids with or without SIS3 treatment. The results showed that in both groups of spheroids, P-Smad3 was primarily present in the nucleus, indicating activation of the TGF-β/Smad signaling pathway. Treatment with SIS3 suppressed the nuclear accumulation of P-Smad3, while Smad3 proteins were predominantly present in the cytoplasm (Fig. 5). These findings suggest that 3D spheroid formation activates the TGF-β/Smad signaling pathway in SHED.

Represent images of p-Smad3 nuclear translocation and localization of Smad3, assayed by immunofluorescence confocal microscopy in spheroids. Nuclei, DAPI (blue) and Smads (green). Scale bar: 20 µm.

Fig. 5.Represent images of p-Smad3 nuclear translocation and localization of Smad3, assayed by immunofluorescence confocal microscopy in spheroids. Nuclei, DAPI (blue) and Smads (green). Scale bar: 20 µm.

4. Discussion

MSCs can be used in regenerative medicine and tissue engineering as a cell-based therapy for treating various clinical conditions, including age-related orthopedic degenerative diseases and autoimmune and inflammatory diseases [59, 60]. MSCs expressing pluripotency markers of ESCs, such as OCT4, NANOG and SOX2, make them promising candidates for clinical applications in tissue regeneration medicine [61]. However, the ex vivo expansion of MSCs can lead to the loss of stem cell properties and a rapid decline in pluripotent gene expression [62].

Recent research suggests that the formation of 3D spheroids enhances and maintains the stemness properties of MSCs [29]. In this study, SHED were cultured in a low attachment 6-well plate and formed compact, round-shaped multicellular aggregates 5 days after seeding. The expression of pluripotent genes (OCT4, SOX2 and NANOG) in SHED increased over time as they were cultured in spheroids for 5 days. Although the exact molecular mechanisms responsible for the upregulation of pluripotent genes in SHED spheroids are not fully understood and remain an area of active research, several theories explain the alterations in the epigenetic status of pluripotent genes in SHED under 3D spheroid culture conditions. Due to reduced oxygen and nutrient supply, the inside of spheroids may become apoptotic and hypoxic [49, 63]. Yamamoto et al. [64] reported that hypoxic and apoptotic cells were present in the center of 3D spheroid aggregations. Hypoxia-inducible factors (HIFs) are key transcription factors of hypoxia-associated genes. Studies have shown that spheroids from mouse MSCs have higher expression of hypoxia-inducible factor-2α (HIF-2α), which plays a critical role in influencing the pluripotency of MSCs [63, 65]. The hypoxic microenvironment in the inner layers of 3D spheroids has been suggested to promote stemness properties by restoring MSCs to a more primitive state [66]. Furthermore, hypoxia has been shown to stimulate the release of extracellular vesicles by human MSCs [67, 68]. For instance, MSC spheroids exhibited higher levels of 370, miR-433 and miR-489, which are responsible for maintaining the quiescent state of adult stem cells [54, 69, 70]. Additionally, SHED spheroids were formed on non-adherent culture conditions that provide lower cytoskeletal tension of cells, contributing to the preservation or enrichment of stemness in MSCs [71, 72].

The 3D spheroid culture of SHED creates a cellular niche that more closely mimics the in vivo microenvironment [39]. The cellular niche of spheroids is presented in the ECM, facilitating cell-cell or cell-ECM interactions much more accessible than in monolayers [49]. This spheroid environment in the niche can help maintain stem cell quiescence, prevent differentiation and downregulate cell cycle progression genes [73]. Moreover, the spheroid culture of MSCs leads to a significant increase in ECM secretion, which is a heterogeneous network of proteins and polysaccharides that transmit physical and biological signals and can control stem cell fate [74, 75]. More importantly, besides determining specific lineage differentiation, ECM serves as a reservoir of growth factors with paracrine activity, playing an essential role in the beneficial effects of MSCs [39, 76, 77]. Paracrine secretion is one of the important mechanisms for the therapeutic application of MSCs in regenerative medicine and tissue engineering. Cytokines produced by MSCs in 3D spheroids exhibit multiple beneficial functions [78]. The 3D spheroid culture of MSCs promotes the paracrine secretion of therapeutic cytokines such as anti-inflammatory protein TSG-6 and angiogenic factors (VEGF and basic fibroblast growth factor (bFGF)), pro-inflammatory cytokines interleukin 1A (IL1A)/IL-1α, interleukin-1β (IL1B)/IL-1β, and interleukin 8 (IL8), anit-apoptotic factors (epidermal growth factor (EGF) and hepatocyte growth factor (HGF)), and the anti-oxidative factor IGF [12, 40]. As members of the transforming growth factors, the TGF-β signaling family controls the expression and activities of key transcription factors closely involved in the modulation of stem cell fate [79, 80]. Our study showed that 3D spheroid formation significantly increased the secretion and mRNA levels of TGF-β, as well as activated the TGF-β/Smad signaling pathway in SHED. The increased immunomodulatory activity of human MSCs in spheroids may be attributed to enhanced intercellular adhesions and cell contact-dependent signaling, which have also been associated with the heightened paracrine activity of MSC aggregates [81, 82].

TGF-β/Smad signals are known to play a crucial role in maintaining the pluripotent state of embryonic stem cells (ESCs) by regulating the expression of stem cell-specific markers [83]. In this study, we demonstrate that 3D spheroid formation induces upregulation of P-Smad3 expression in SHED and increases the expression levels of OCT4, NANOG and SOX2, primarily through the canonical Smad3 signaling pathway. Although the precise mechanism underlying the activation of TGF-β/Smad signaling by these interactions is unclear, the results suggest that TGF-β/Smad signaling involves a priming process that enhances the stemness properties of SHED in spheroids. TGF-β-mediated phosphorylation of Smad3 on spheroids is essential for TGF-β/Smad signal transduction. Previous studies have reported that Smads can bind to the promoter of pluripotent genes and regulate their expression [52]. However, it is uncertain whether the binding of Smads to target genes occurs through the formation of complexes with other transcription factors, and the functional significance of any competition between SMAD2/3 and Smad1/5/8 for the cofactor Smad4 remains unclear.

5. Conclusions

In summary, this study investigated the regulation of stemness in SHED cultured in 3D spheroids. The results revealed that SHED spheroids activated the TGF-β/Smad signaling pathway and enhanced the stemness properties of SHED. The higher levels of TGF-β produced by SHED spheroids may contribute to cellular transduction and the multiple beneficial functions of MSCs, thereby indicating that MSC spheroids might have promising therapeutic applications in regenerative medicine and tissue engineering.

Availability of data and materials

Not applicable.

Author contributions

JX and HWL—designed the research study. WBW and XMS—performed the research. HYK, SQQ, XHZ, QHZ, XHY and XTZ—analyzed the data. JX and JJ—wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

SHEDs were isolated from patients after obtaining informed consent from their parents at the department of pediatric dentistry of Shenzhen Longgang E.N.T. Hospital (Shenzhen, China). The study was approved by the Ethics Committee of Shenzhen Longgang E.N.T. Hospital (Reference number of approval of the Ethics Committee: ZSSOM No. 2021-0130).

Acknowledgment

Not applicable.

Funding

This work was supported by the Natural Science Foundation of Guangdong Province, China (General Program: 2020A1515010237); Shenzhen Key Medical Discipline Construction Fund (No. SZXK039); Shenzhen Science and Technology Innovation Commission, China (Free Exploration Projects: JCYJ20180305163353862 and JCYJ20180305163259711); Special Fund for Science and Technology Development of Longgang District, Shenzhen (LGKCYLWS2021000031 and LGKCYLWS2019000656); and The Key Medical Talents Training Project of Xuzhou (XWRCHT20220060).

Conflict of interest

The authors declare no conflict of interest.

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