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Epithelial differentiation of gingival mesenchymal stem cells enhances re-epithelialization for full-thickness cutaneous wound healing

Stem Cell Research & Therapy volume 15, Article number: 455 (2024) Cite this article

Abstract

Background

Increasing evidence suggests that mesenchymal stem cells (MSCs) repair traumatized tissues primarily through paracrine secretion and differentiation into specific cell types. However, the role of epithelial differentiation of MSCs in cutaneous wound healing is unclear. This study aimed to investigate the epithelial differentiation potential of gingival tissue-derived MSCs (GMSCs) in epithelial cell growth medium and the mechanisms underlying their differentiation into an epithelial-like cell phenotype.

Methods

We used scanning electron microscopy to examine GMSCs for epithelial differentiation. Quantitative real-time polymerase chain reaction and Western blotting were respectively used to measure genes and proteins related to epithelial differentiation. Immunofluorescence was used to examine subcellular localization of KLF4, KRT19, and β-catenin proteins. Transcriptome sequencing was used to enrich the mechanisms underlying epithelial differentiation in GMSCs. An MSAB inhibitor was used to validate the Wnt signaling pathway further. The wound healing rate and re-epithelialization were assessed through macroscopical observation and hematoxylin and eosin staining.

Results

GMSCs cultured in epithelial cell growth medium from days 3 to 15 exhibited decreased expression of mesenchymal-epithelial transition and stemness-related proteins (N-cadherin, Vimentin, KLF4, and SOX2), increased expression of epithelial-related proteins (KRT12, KRT15, KRT19, and E-cadherin), and exhibited epithelial-like morphology. Mechanistically, high-throughput sequencing revealed that the Wnt and TGF-beta signaling pathways were inhibited during epithelial differentiation of GMSCs (Epi-GMSCs). MSAB-induced Wnt signaling pathway inhibition promoted epithelial-related gene and protein expression. Furthermore, we demonstrated the ability of Epi-GMSCs to facilitate wound healing by improving re-epithelialization in a full-thickness skin defect model.

Conclusions

Collectively, this study uncovers that GMSCs have the ability to differentiate into epithelia and highlights a promising strategy for using Epi-GMSCs to improve cutaneous wound healing.

Introduction

The skin is the largest organ of the human body, which functions as the primary barrier against external threats. The skin in the maxillofacial region is particularly vulnerable to trauma, burns, infections, tumors, and other harmful factors that compromise its protective functions. Furthermore, defects in the maxillofacial skin have significant implications for the aesthetics, speech, and mental health of an individual. Therefore, the restoration and regeneration of maxillofacial tissue after such defects is a significant clinical concern. Traditional skin substitutes are hindered by poor vascularization, microbial contamination, and severe postoperative scarring [1].

Recent years have witnessed substantial interest in employing mesenchymal stem cell (MSC) therapy to enhance the healing process in acute and chronic wounds because of their ability to differentiate into various cell types, including osteoblasts, chondrocytes, adipocytes, endothelial cells, and keratinocytes [2, 3]. Additionally, MSC transplantation can regulate immune function and enhance angiogenesis [4, 5]. Wound healing is a complex biological process that includes immune cell migration, angiogenesis, granulation tissue formation, re-epithelialization, and extracellular matrix remodeling [6]. During these phases, re-epithelialization is essential in restoring the barrier function of the skin during the healing of epidermal wounds [7, 8]. MSCs accelerate wound healing through several mechanisms, including enhanced angiogenesis and differentiation into endothelial cells through proangiogenic factor secretion, extracellular matrix synthesis, M2 macrophage polarization, endogenous MSC recruitment, and apoptotic body generation [9, 10]. However, during in vivo skin defect treatment, the ability and mechanism of epithelial differentiation of MSCs are unclear.

Over the past decade, numerous studies reported that MSCs and pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) can differentiate into corneal epithelial cells following in vitro induction [11,12,13,14,15,16]. The expression of keratin (KRT)-related genes, including KRT3, KRT8, KRT12, KRT14, KRT15, and KRT19, increased to varying degrees in stem cells after induction of epithelialization. KRT is an intermediate filament protein of the cytoskeleton that serves as a marker for epithelial cell growth, differentiation, and maturation and provides structural support for epithelial cells [17, 18]. Furthermore, KRT is essential in maintaining the continuity and integrity of the epithelium during wound healing [19]. Recent studies reported that KRT hydrogel in wound dressing significantly enhances dermal repair and regeneration in a full-thickness excision wound model [20, 21]. However, the potential therapeutic effects of epithelium-differentiated MSC transplantation in the repair of skin defects are unknown.

Therefore, the goal of the present study was twofold (Fig. 1). The first aim was to clarify the mechanism of epithelial differentiation of gingival tissue-derived MSCs (GMSCs) using high-throughput sequencing technology. The second aim was to examine whether epithelial differentiation of GMSCs (Epi-GMSCs) could promote re-epithelialization in vivo.

Fig. 1
figure 1

Schematic illustration of the experiment design. (A) Schematic diagram illustrating the mechanism of epithelial differentiation of GMSCs. (B) Schematic diagram illustrating the role of Porous GelMA encapsulating Epi-GMSCs in promoting re-epithelialization and wound healing

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Materials and methods

Antibodies and reagents

Rabbit monoclonal antibodies against IgG (ab172730), CD34 (ab81289), CD73 (ab227030), CD105 (ab231774), KRT19 (ab52625), SOX2 (ab92494), and RUNX2 (ab92336) were procured from Abcam. Mouse monoclonal antibodies against CD90 (ab11155) were obtained from Abcam. Rabbit polyclonal antibodies against CD45 (ab10558) were acquired from Abcam. Rabbit monoclonal antibodies against Vimentin (5741), COL1A1 (39952), and LRP5 (5731) were procured from Cell Signaling Technology. Rabbit polyclonal antibodies against KRT12 (A9642) and KRT15 (A4854) were obtained from ABclonal. Rabbit polyclonal antibodies against E-cadherin (20874-AP), N-cadherin (22018-1-AP), KLF4 (11880-1-AP), and GAPDH (10494-1-AP) were received from Proteintech. Mouse monoclonal antibody against β-catenin (66379-1-Ig) was obtained from Proteintech. The complete medium of human gingival epithelial cells (GECCM) was sourced from Wuhan Pricella Biotechnology Co., Ltd. The primary constituent of the complete medium was Dulbecco’s modified Eagle’s medium-F12, which included 2% fetal bovine serum and 1% penicillin-streptomycin, supplemented with an epithelial cell growth supplement (epithelial growth factor, insulin, hydrocortisone, epinephrine, thyroxine, transferrin, and selenium solution). MSAB (Cat. No. HY-120697, Wnt/β-catenin pathway inhibitor) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Human immortalized gingival epithelial cells (GECs) were acquired from Bluef (Shanghai) Biotechnology Development Co., Ltd. Human GMSCs were kindly donated by Dr. Zhentian Xu of Zhejiang University. GMSCs were isolated and cultured as described in our previous study [22].

Flow cytometry

Flow cytometry was performed to evaluate the expression of surface markers on GMSCs. A 40 mm cell strainer was used to digest and filter GMSCs, which were subsequently washed with cold FACS washing buffer and incubated for 30 min at 4 °C with primary antibodies: IgG, CD34, CD45, CD73, CD90, and CD105.

Cell culture and epithelial induction

GMSCs from passages 2–4 were suspended in a complete medium and inoculated in six -well plates at a density of 2 × 105 cells/well. GECCM was added after 24 h. The morphological changes of GMSCs were examined at various time intervals using an inverted phase-contrast microscope.

Scanning electron microscopy (SEM)

SEM was performed as previously described to examine the GMSCs morphology and changes on the cell surface at a higher magnification and greater depth than feasible with optical microscopy. Briefly, GMSCs were seeded on 14 mm coverslips and cultured overnight. The samples were initially fixed with 2.5% glutaraldehyde and postfixed in 1% osmium acid for SEM analysis on days 3, 9, and 15 after GECCM induction. After dehydration with gradient alcohol and gold spraying, it was observed using an FEI Nova Nano SEM 450.

cDNA synthesis and quantitative real-time PCR (RT-qPCR)

For total RNA extraction, cell samples induced using GECCM for days 3, 9, and 15 were lysed with TRIzol (Invitrogen, USA). Total cDNA synthesis was performed using the One Step PrimeScript miRNA cDNA Synthesis Kit (Takara, Japan). RT-qPCR was performed using SYBR green (Takara, Japan) and a CFX384 Real-Time PCR Detection System (Bio-Rad, USA). The relative expression level of the target genes was calculated using the 2−ΔΔCt method, and the endogenous GAPDH gene was used as the control. Specific primer sequences were listed in Supplementary Table 1.

Western blot (WB) analysis

WB analysis was performed to determine protein expression levels. Total protein lysates were extracted using a radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (EpiZyme, China). Total proteins were separated using SDS-PAGE (7.5% or 12.5% gel), transferred to PVDF membranes, and blocked with 5% nonfat dry milk in TBST for 2 h at room temperature. The blots were incubated overnight at 4 °C with the corresponding primary antibody. Finally, protein bands were detected using chemiluminescent detection reagents (EpiZyme, China).

Immunofluorescence (IF)

IF was performed to identify changes in the cellular sub-localization of proteins following GECCM induction. GMSCs were incubated overnight on 14 mm coverslips. After GECCM induction, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked using an Immunol Staining Blocking Buffer (Beyotime, China). The corresponding fluorescence secondary antibodies were incubated at room temperature for 1 h following the overnight incubation of primary antibodies at 4 °C in a humidified chamber. Subsequently, the samples were observed and photographed under a laser scanning confocal microscope (Nikon, Japan).

RNA-seq

Cell samples were extracted with the TRIzol reagent to obtain total RNA, which was stored in cryopreservation tubes at -80 °C for subsequent sequencing analysis. All sequencing and data analysis work was completed by the Biomarker Technologies (BMK, China).

Preparation of porous GelMA

The Porous GelMA was prepared following the method used in our previous study [22]. Briefly, the Porous GelMA (GM-PR, EFL-GM-PR-002, EFL, Suzhou, China) was dissolved in a phosphate-buffered saline solution to obtain an 8% (w/v) concentration, which was stirred for 1 h at 37 °C. Based on the manufacturer’s protocol, GM-PR hydrogels were mixed with GMSCs or Epi-GMSCs.

Cutaneous wound healing experiments

A total of 20 male Balb/c nude mice (6 weeks, 20 g) were purchased from Slack Laboratory Animal Company (Shanghai, China). The animals were kept at 22–25 °C with 50-60% humidity and a 12-h light-dark cycle in the Animal Center of Zhejiang University. Animals were housed in standard cages with five animals per cage. This study was approved by the Ethics Committee of Zhejiang University (Approval No. ZJU20230339). We anesthetized the mice using 2.0% isoflurane via inhalation and maintained them under anesthesia with 1.0% isoflurane/oxygen. We created full-thickness circular skin wounds (10 mm in diameter) on the back of the mice. Subsequently, the mice were randomly divided into blank, GM-PR, GM-PR + GMSCs, and GM-PR + Epi-GMSCs groups, with five mice in each group. We locally injected 100 µL (1 × 106 cells) of the cell-loaded hydrogels using a pipette gun to cover the wounds with full skin defects and subsequently photocured after crosslinking with 405 nm blue light for 10 s. Antibiotics were administered intramuscularly for three days to prevent postoperative infection. The dressing was not changed during the treatment due to scabbing. The wound healing was regularly observed and photographed on days 1, 4, 8, and 12. The formula below was used to calculate the wound healing rate (%):

$${\text{Wound}}\:{\text{healing}}\:{\text{rate}}\left( \% \right) = \frac{{{S_1} - {S_t}}}{{{S_1}}} \times 100\%$$

S1 denotes the initial wound area (day 1), and St denotes the wound healing subsequent times, specifically days 4, 8, and 12. Image J software (NIH, Bethesda, MD, USA) was used to measure the wound healing area. The animals all exhibited similar strain, age, and sex, and the data were analyzed using a single-masked approach. Two researchers examined each animal: one who randomly performed the surgical (the only one aware of the treatment group assignment) and another who collected and analyzed the data. Animals were excluded from this study if they died before the conclusion of the experiment. However, no samples were excluded during the data analysis. This study was conducted in accordance with the ARRIVE guidelines 2.0.

Histology and image acquisition

Healed skin samples were collected on day 12 and fixed in paraformaldehyde. After sample collection, the animals were euthanized using an overdose of isoflurane and cervical dislocation while their heart rate and respiratory rate were monitored. Paraffin-embedded sections of the samples were prepared at 4 μm per slide and stained with hematoxylin-eosin (HE) staining. Histological changes were observed and photographed using a Leica DM4000 upright microscope (Leica Microsystems, Germany).

Statistical analysis

Each experiment was repeated at least thrice, and all experiments were statistically analyzed in triplicate. GraphPad Prism software (version 9) was used for data analysis. One-way analysis of variance was used to compare statistical differences among multiple groups. Data are presented as mean ± standard deviation. Significant difference was defined by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

GMSCs exhibited epithelial-like morphology when cultured in GECCM

GMSCs were 99.96%, 99.99%, and 100% positive for MSC-associated markers CD73, CD90, and CD105, respectively, while exhibiting 0.31% and 0.35% positive for hematopoietic stem cell markers, CD34 and CD45 (Supplementary Fig. 1). The morphological changes of GMSCs in GECCM prior to assessing their epithelial differentiation potential. As the culture duration increased, the morphology of the GMSCs gradually changed from a shuttle shape to an irregular polygonal form, exhibiting epithelial-like characteristics (Fig. 2A). SEM was used to directly observe the ultrastructural changes of differentiated GMSCs to further confirm the effects of GECCM on epithelial differentiation in GMSCs. As shown in Fig. 2B, GECCM-induced GMSCs on culture days 3, 9, and 15 exhibited a large number of irregular protrusions, whereas GMSCs displayed a relatively smooth cell surface on day 0. These irregular protrusions resemble those on the surface of other epithelial cells called microcilia. These results revealed that GECCM-induced GMSCs undergo epithelial differentiation.

Fig. 2
figure 2

GMSCs exhibited epithelial-like morphology when cultured in GECCM. (A) Representative images of GMSCs captured under an inverted phase contrast microscope after days 3, 9, and 15 of incubation in GECCM. (n = 3). (B) Representative SEM images of GMSCs after days 3, 9, and 15 of incubation in GECCM. (n = 3)

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Epi-GMSCs exhibited decreased expression of mesenchymal-epithelial transition (MET) and stemness-related genes and proteins and increased expression of epithelial-related genes and proteins

MET, stemness, and epithelial-related genes and proteins were examined to further validate our findings. The prolongation of GECCM induction culture duration resulted in a significant decrease in the expression of N-cadherin, Vimentin, KLF4, SOX2, OCT4, and NANOG genes on day 0 (Fig. 3A). Consistent with gene expression, the protein expression levels of N-cadherin, Vimentin, KLF4, and SOX2 were markedly decreased (Fig. 3B and C). We further conducted immunofluorescent staining to evaluate the protein expression and localization of KLF4. Quantitative fluorescent intensity showed a significant decrease in KLF4 immunoreactivity after treatment with GECCM for 15 days (Fig. 3D). As shown in Fig. 3E, a striking reduction in the fluorescence of KLF4 protein in the nucleus was found after treatment with GECCM for 15 days.

Fig. 3
figure 3

The expression levels of MET and stemness-related genes and proteins were decreased in Epi-GMSCs. (A) RT-qPCR analysis of the expression of N-cadherin, Vimentin, KLF4, SOX2, OCT4, and NANOG after days 3, 9, and 15 of incubation in GECCM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (B) WB and (C) quantitative analysis of the expression of N-cadherin, Vimentin, KLF4, and SOX2 after days 3, 9, and 15 of incubation in GECCM. Full-length blots/gels are presented in Supplementary Fig. 2. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). (D) Quantitative analysis of KLF4 immunofluorescence intensity using ImageJ. (n = 3, **P < 0.01). (E) Representative confocal fluorescence images showing the subcellular localization of KLF4 (red). Cytoskeleton analysis was performed by immunofluorescence staining with F-actin (green). (n = 3)

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However, the expression levels of epithelial-related genes and proteins were elevated in Epi-GMSCs. The mRNA expression levels of KRT12, KRT15, KRT19, KRTAP2-3, and ITGB3 were significantly higher on days 3, 9, and 15 than on day 0 (Fig. 4A). Similarly, the protein expression levels of KRT12, KRT15, KRT19, and E-cadherin coincidentally increased after GECCM treatment (Fig. 4B and C). Furthermore, we focused on the intracellular localization of KRT19 in GMSCs using IF cytochemistry and used GECs as a positive control. As shown in Fig. 4D, the fluorescence intensity of the KRT19 protein, expressed predominantly in the cell membrane and cytoplasm, was markedly increased after GECCM induction. Therefore, these results indicated that Epi-GMSCs gradually underwent epithelial differentiation with the extension of culture time.

Fig. 4
figure 4

The expression levels of epithelial-related genes and proteins were elevated in Epi-GMSCs. (A) RT-qPCR analysis of the expression of KRT12, KRT15, KRT19, KRTAP2-3, and ITGB3 after days 3, 9, and 15 of incubation in GECCM (n = 3, *P < 0.05, **P < 0.01, ****P < 0.0001). (B) WB and (C) quantitative analysis of the expression of KRT12, KRT15, KRT19, and E-cadherin after days 3, 9, and 15 of incubation in GECCM. Full-length blots/gels are presented in Supplementary Fig. 3. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). (D) Representative confocal fluorescence images showing the subcellular localization of KRT19 (red, white arrows). Cytoskeleton analysis was performed by immunofluorescence staining with F-actin (green). GECs served as a positive control for high KRT19 protein expression. (n = 3)

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RNA-Seq analysis revealed significantly decreased wnt signaling components in Epi-GMSCs

This study used RNA-Seq to investigate the molecular mechanism by which GECCM enhanced epithelial-related gene and protein expression. The volcano plot revealed 1,047 upregulated and 1,427 downregulated genes in Epi-GMSCs (Fig. 5A). The heatmap below indicates that genes involved in Wnt and TGF-beta signaling pathways were significantly downregulated in Epi-GMSCs (Fig. 5B). The findings of GO (molecular function) and KEGG functional enrichment analysis were consistent with the above description. The Wnt signaling pathway was simultaneously enriched (Fig. 5C and D).

Fig. 5
figure 5

RNA-Seq analysis revealed significantly decreased Wnt signaling components and TGF-beta signaling pathway in Epi-GMSCs. (A) Volcano plot indicating the total number of genes differentially up-regulated and down-regulated. (B) Heat map generated from gene expression array of Wnt signaling pathway and TGF-beta signaling pathway-related genes. (C) GO and (D) KEGG analysis of RNA-Seq data showing downregulation of Wnt signaling pathway in GMSCs after 9 days of incubation in GECCM. (n = 3)

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Validation of the RNA-Seq results, revealed that Epi-GMSCs decreased the expression of Wnt signaling pathway-related genes and proteins. RT-qPCR revealed that the mRNA expression levels in the validated genes were significantly reduced on days 3, 9, and 15 than on day 0 (Fig. 6A). Additionally, the WB findings were consistent with those of the RT-qPCR (Fig. 6B and C). Furthermore, GECCM induction markedly reduced the fluorescence intensity of β-catenin protein in the cell membrane and cytoplasm (Fig. 6D and E). We examined signaling pathways including Notch, Hippo, and JAK-STAT, which are essential in cell differentiation, and found that genes associated with these pathways were significantly down-regulated on days 3, 9, and 15 than on day 0 (Supplementary Figs. 5–7). These results suggest that cell differentiation results from multiple signaling pathways involved in regulation.

Fig. 6
figure 6

The expression levels of Wnt signaling pathway-related genes and proteins were decreased in Epi-GMSCs. (A) RT-qPCR analysis of the expression of LRP5, LRP6, COL1A1, β-catenin, RUNX2, WNT3, WNT5A, WNT11, FZD5, FZD7, and TCF7L2 after days 3, 9, and 15 of incubation in GECCM (n = 3, ****P < 0.0001). (B) WB and (C) quantitative analysis of the expression of LRP5, COL1A1, β-catenin, and RUNX2 after days 3, 9, and 15 of incubation in GECCM. Full-length blots/gels are presented in Supplementary Fig. 4. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (D) Quantitative analysis of β-catenin immunofluorescence intensity using ImageJ. (n = 3, *P < 0.05). (E) Representative confocal fluorescence images showing the subcellular localization of β-catenin (red). Cytoskeleton analysis was performed by immunofluorescence staining with F-actin (green). (n = 3)

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We further validated the role of the Wnt signaling pathway in epithelial differentiation using the Wnt/β-catenin pathway inhibitor MSAB. As shown in Fig. 7A, the cell morphology changed from single, long spindle-shaped cells to a cobblestone-like epithelium shape after a 3-day treatment with MSAB and GECCM. The inhibitor MSAB binds to β-catenin protein, resulting in its degradation and thus downregulating Wnt pathway target genes and proteins. As expected, RT-qPCR revealed no significant changes in the level of β-catenin mRNA (Fig. 7B), but the WB indicated that the protein level of β-catenin in the GECCM + MSAB group was significantly down-regulated (Fig. 7C and D). Compared with the GECCM group, the mRNA expression levels of KRT12, KRT15, and KRT19 were significantly up-regulated after treatment with MSAB (Fig. 7E). Meanwhile, RT-qPCR revealed that the expression levels of genes associated with the Wnt signaling pathway were further decreased after MSAB treatment (Fig. 7E).

Fig. 7
figure 7

Inhibition of the Wnt signaling pathway promoted the expression of epithelial-related genes. (A) Representative images of GMSCs captured under an inverted phase contrast microscope after 3 days of incubation in GECCM with and without MSAB (5 µM) (n = 3). (B) RT-qPCR and (C, D) WB analysis of the expression of β-catenin after 3 days of incubation in GECCM with and without MSAB (5 µM) (n = 3, nsP > 0.05, *P < 0.05). Full-length blots/gels are presented in Supplementary Fig. 8. (E) RT-qPCR analysis of the expression of KRT12, KRT15, KRT19, LRP5, RUNX2, and COL1A1 after 3 days of incubation in GECCM with and without MSAB (5 µM) (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

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GM-PR hydrogels loaded with Epi-GMSCs accelerated the healing of full-thickness wounds in mice

The potential biological functions of Epi-GMSCs in vivo were further examined using a full-thickness skin defect model and a series of experiments (Fig. 8A). We prepared GM-PR hydrogel, GMSCs + GM-PR hydrogel, and Epi-GMSCs + GM-PR hydrogel to treat the full-thickness wounds. During the 12 days, the wound areas in all four groups gradually decreased (Fig. 8B). Statistical analysis revealed that the Epi-GMSCs + GM-PR group exhibited the fastest wound healing rate than the other groups (Fig. 8C). The Epi-GMSCs + GM-PR group exhibited a statistically significant higher wound healing rate than the GM-PR and control groups throughout the wound healing process (Fig. 8C). The wound healing rate of the Epi-GMSCs + GM-PR group was significantly higher than that of the GMSCs + GM-PR group after 8 days of treatment (Fig. 8C). These results indicated that Epi-GMSCs exhibited a beneficial effect on wound healing during the early stages. We used HE stains on the wound tissue in regenerated skin to further evaluate its histological structure. On day 12, the Epi-GMSCs + GM-PR group exhibited thinner and denser epidermis and neogenic hair follicles than other groups (Fig. 8D). Furthermore, the Epi-GMSCs + GM-PR group exhibited reduced neutrophil infiltration in the connective tissue, suggesting that wound tissue reconstruction and re-epithelialization processes were facilitated (Fig. 8D).

Fig. 8
figure 8

GM-PR hydrogel loaded with Epi-GMSCs accelerated the healing of full-thickness wounds in mice. (A) Schematic diagram depicting the whole experimental procedure in vivo. (B) Representative photographs of wound healing assays with different dressings on days 1, 4, 8, and 12. (n = 5). (C) Quantitative analysis of the wound healing rate at different time points (n = 5, *P < 0.05, **P < 0.01, ****P < 0.0001). (D) The representative HE staining pictures of wound tissues from four groups on day 12 (yellow dotted line: epidermis). (n = 5)

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Discussion

MSC-based therapy is essential in tissue engineering and regenerative medicine because of its multidirectional differentiation potential. The mechanisms underlying epithelial differentiation towards MSCs and their role in cutaneous wound healing are unknown. This study examined the morphology of Epi-GMSCs and the expression of genes and proteins associated with epithelial and mesenchymal cells. Epi-GMSCs exhibited epithelial-like cell morphologies and inhibited the Wnt signaling pathway, promoting the expression of epithelial-associated genes and proteins. Experiments in a complete skin defect model demonstrated the ability of Epi-GMSCs to promote re-epithelialization and accelerate healing.

Several organs and tissues in the body contain tissue-specific stem cells, which can differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, vascular endothelial cells, neural cells, hepatocytes, and corneal epithelial cells [23]. However, uncertainty persists regarding the mechanism and function of MSC differentiation into epithelial cells during the repair of skin defects. Considering the factors including easy accessibility, minimal trauma in the donor region, easy expansion in vitro, and enhanced immunomodulatory ability [24], we selected GMSCs as the progenitor cells for epithelial differentiation.

Cellular plasticity is a fundamental feature of numerous biological processes and disease. The transformation of mesenchymal cells into epithelial cells begins at an early stage of embryonic development, and MET is essential for organ damage repair [25]. These findings enable the investigation of epithelial differentiation of MSCs in vitro. After one week of induction with collagen IV and fibroblast-conditioned medium, Ahmad et al. reported that epithelial differentiated human embryonic stem cells possess many microcilia [11]. These structures are similar to our SEM observations with GMSCs, indicating that epithelial differentiation of GMSCs can be successfully induced by epithelial cell growth medium. Consistent with the findings of previous studies [12,13,14,15, 26], we found that MET and stemness-related genes and proteins expression decreased while epithelial-related genes and proteins expression increased during differentiation induction. However, discrepancies existed among the various studies regarding epithelial-related genes and proteins that were up-regulated. These differences may be associated with the type of differentiated MSCs and the conditions of their induction. As a result, future studies should focus on inducing MSCs to highly express certain epithelial-associated proteins, which are crucial for tissue engineering and regenerative medicine.

High-throughput sequencing technologies have gradually become effective tools for studying molecular mechanisms as sequencing technology advances. Herein, RNA-seq revealed that Wnt and TGF-beta signaling pathways were inhibited during GMSC epithelial differentiation. Consistent with our findings, two previous studies reported that XAV939 [27] or DKK1 [28], inhibitors of Wnt/β-catenin signaling, facilitated the differentiation of bone marrow-derived MSCs into an epithelial-like phenotype when co-cultured with alveolar epithelial cells. Furthermore, another previous study demonstrated that human adipose-derived MSCs can transition to epithelial lineage by inhibiting GSK3 and TGF-beta signaling pathways [29]. Accordingly, the epithelial differentiation of MSCs is a complex process that seemingly involves multiple signaling pathways.

Wound healing is a complex physiological process involving the coordinated action of several different tissues and cell lineages, primarily including keratinocytes, epidermal stem cells, fibroblasts, adipocytes, and endothelial cells [30]. Keratinocytes specifically secrete various cytokines and KRTs that regulate wound re-epithelialization [31]. KRT performs multiple functions in skin cells, including providing mechanical support and damage prevention and actively participating in every stage of wound healing [32,33,34]. Previous studies reported that in vitro and in vivo experiments revealed that KRT-based wound dressings accelerate wound healing and tissue regeneration [35, 36]. Besides, KRT is significantly abundant in epithelial-differentiated MSCs, and the transplantation of these cells has demonstrated efficacy in enhancing corneal epithelial regeneration [37]. The cutaneous wound healing process is similar to that of corneal epithelial repair. This study confirmed that Epi-GMSCs expressed KRT12, KRT15, KRT19, and KRTAP2-3 and could facilitate cutaneous wound healing by accelerating re-epithelialization.

This study has some limitations. First, it failed to utilize multiple cell types to validate its results. Second, the underlying mechanisms of Epi-GMSCs have not been sufficiently explored. Future forward and reverse experimental validation is required. Additionally, the effectiveness and applicability of the Epi-GMSCs should be verified in other laboratory animals, including rats and miniature pigs.

Conclusions

We demonstrated that GMSCs cultured in an epithelial cell growth medium facilitate the mesenchymal-to-epithelial transition process by inhibiting the Wnt signaling pathway. Additionally, we provided evidence that Epi-GMSCs can enhance wound healing through improved re-epithelialization. These findings contribute to a better understanding of the Epi-GMSCs, indicating their significant potential as therapeutic agents.

Data availability

Targeted high-throughput sequencing data has been submitted to the Sequence Read Archive (SRA) under the SRA accession number SRP524665 and BioProject accession number PRJNA1145301. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

MSCs:

Mesenchymal stem cells

GMSCs:

Gingival tissue-derived MSCs

Epi-GMSCs:

Epithelial differentiation of GMSCs

GECCM:

Complete medium of human gingival epithelial cells

GECs:

Human immortalized gingival epithelial cells

SEM:

Scanning electron microscopy

RT-qPCR:

Quantitative real-time PCR

WB:

Western blot

IF:

Immunofluorescence

HE:

Hematoxylin-eosin

MET:

Mesenchymal-epithelial transition

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Acknowledgements

We are very grateful to Dr. Zhentian Xu for donating GMSCs. We thank Dandan Song at the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for her technical assistance on scanning electron microscopy. Figures 1 and 7A were created with BioRender.com. We thank Home for Researchers editorial team (www.home-for-researchers.com) for language editing service.

Funding

This work was supported by the Postdoctoral Fellowship Program of CPSF under Grant Number GZC20232290, the National Natural Science Foundation of China (Grant No. 82370928, Grant No. 82271001), and the Major Project of the Science and Technology Department of Zhejiang Province (Grant No. 2021C03113).

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Authors and Affiliations

  1. Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou, 310000, China

    Yongzheng Li, Lingling Dong, Yani Chen, Wenjin Cai, Guoli Yang & Ying Wang

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  1. Yongzheng Li
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Contributions

Y.L. and G.Y. conceived the idea and designed the biological experiments. L.D., W.C. and Y.C. contributed to data quantification and performed statistical analysis. Y.L. wrote the paper and all authors commented on the manuscript. G.Y. and Y.W. revised it critically. Y.W. supervised the project.

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Correspondence to Guoli Yang or Ying Wang.

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Ethics approval and consent to participate

The original sources (Bluef Biotechnology Development Co., Ltd. and Dr. Zhentian Xu’s lab) have confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent. The animal study protocol was approved by the Ethics Committee of Zhejiang University (Title of the approved project: Epithelial differentiation of gingival mesenchymal stem cells enhances re-epithelialization for full-thickness cutaneous wound healing; Approval No: ZJU20230339; Date of approval: 2023.09.06) and conducted in accordance with the ARRIVE (Animal Research: Reporting of In vivo Experiments) guidelines 2.0.

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All authors agreed to the publication of this paper.

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The authors declare that they have no competing interests.

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Li, Y., Dong, L., Chen, Y. et al. Epithelial differentiation of gingival mesenchymal stem cells enhances re-epithelialization for full-thickness cutaneous wound healing. Stem Cell Res Ther 15, 455 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04081-9

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512