hESC-derived mesenchymal stem cells Promote Oral Mucositis Healing via the PI3K/AKT Pathway

hESC-derived mesenchymal stem cells Promote Oral Mucositis Healing via the PI3K/AKT Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article hESC-derived mesenchymal stem cells Promote Oral Mucositis Healing via the PI3K/AKT Pathway Kejia Lv, Bicong Gao, Chenlu Shen, Weijia Ye, Yanan Yao, Hua Yao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3449081/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Oral mucositis (OM) is among the most pervasive adverse reactions caused by radiotherapy or chemotherapy during cancer treatment. This study focused on the reparative effects of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) in OM and possible mechanisms. Materials and Methods An ulcer model was created in the rat buccal mucosa to simulate OM, and hESC-MSCs were injected 48h later to assess their reparative effects. The efficacy of hESC-MSCs in regulating apoptosis and proliferation in LPS- or 5-FU-injured HaCaT cells was studied in vitro using a transwell coculture system. Subsequently, the PI3K inhibitor LY24002 was used to assess whether hESC-MSCs regulated injured HaCaT cells through PI3K/AKT pathway. Results We found that hESC-MSCs injection promoted OM healing in rats through the acceleration of re-epithelialization, and a decrease in apoptosis. Our findings also revealed that the hESC-MSCs treatment led to a reduction in the quantity of HaCaT cells undergoing apoptosis. Western blot analysis revealed that hESC-MSCs activated AKT, resulting in increased protein levels of PCNA and BCL-2, decreased protein levels of Bax and Caspase-3. Whereas, LY294002 reversed these changes. Conclusions hESC-MSCs promoted OM healing, inhibited LPS- or 5-FU-injured HaCaT cell apoptosis, and increased their proliferation via the PI3K/AKT pathway. Oral mucositis human embryonic stem cell-derived mesenchymal stem cells Oral mucosal injury PI3K/AKT signaling Transwell coculture system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Oral mucositis (OM) is among the most pervasive adverse reactions caused by radiotherapy or chemotherapy during cancer treatment 1,2 . Chemo/radiotherapy-induced OM primarily manifests as congestion and erosion in the mouth, which is accompanied by a burning sensation and pain, making it difficult for patients to chew and swallow and substantially impairing nutritional intake and quality of life. Despite the terrible clinical effects of OM, patients have few methods to prevent or minimize this condition. In addition, the few therapies validated by high levels of evidence are not always applicable to all types of OM, and their effects on tissues are not fully understood, leading to large obstacles for patients receiving cancer treatment 3 . Therefore, more efficacious therapies and preventive guidelines for treating OM are urgently required in the clinic 4,5 . Mesenchymal stem cells (MSCs), a type of stem cell, have the capacity to mitigate tissue damage resulting from inflammation and promote the healing process after injury and have been used to prevent and treat adverse chemo/radiotherapy reactions 6 . Apart from their ability to regenerate, MSCs can migrate to injured tissues and exert paracrine effects such as the recruitment of endogenous progenitor cells and the production of growth factors that promote tissue regeneration 7 . Several studies have demonstrated the efficacy of MSC therapy in the treatment of chemotherapy/radiotherapy-induced mucositis 5,8,9 . However, different biological properties of MSC populations may affect their therapeutic efficacy in this disease. Another challenge is obtaining enough MSCs and culturing them in vitro 10 . The process of isolating and cultivating human embryonic stem cells (hESCs), which possess the unique ability to undergo unlimited self-renewal and differentiate into various cell types, has introduced novel opportunities for deriving MSCs 11 . hESC-derived MSCs (hESC-MSCs) exhibit enhanced attributes such as a consistent phenotypic, significant proliferative capacity, and consistent immunoregulatory functionality. Consequently, these cells are seen as more appropriate for extensive cultivation compared to MSCs generated from alternative sources. Several studies have recently reported that hESC-MSCs have immunomodulatory and regenerative potential in the treatment of a variety of diseases 10,12–15 . However, no studies have been conducted to investigate their therapeutic effects on OM and their potential mechanism. Therefore, we hypothesized that hESC-MSCs play a beneficial role in OM repair. To validate our hypothesis, we compared the healing rate in a rat mucosal injury OM model, with and without the injection of hESC-MSCs. Following that investigated the mechanism by which hESC-MSCs promote repair in a lipopolysaccharide (LPS)- or 5-fluorouracil (5-FU)-induced HaCaT cell injury model. We discovered that hESC-MSCs promoted OM wound healing by stimulating the proliferation of epithelial cells and inhibiting their apoptosis in rat models. Furthermore, hESC-MSCs mediate the PI3K/AKT pathway to modulate apoptosis injured by LPS or 5-FU in HaCaT cells as well as promote their proliferation. 2. MATERIAL AND METHODS 2.1 Animaland ethical considerations Male Wistar rats (8–10 weeks old and 230–270 g in weight) were obtained from the Zhejiang Academy of Medical Sciences. The Institutional Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine authorized the animal research(NO:20201431). 2.2 hESC-MSCs obtainment and identification hESC-MSCs were purchased from Hangzhou Yuansheng Biotechnological Co., LTD, and cultured with the hESC-MSCs medium (Hangzhou Yuansheng Biotechnological Co., LTD., China). Two steps were used to separate hESC-MSCs from hESCs. Briefly, H9-ESC colonies (Hangzhou Yuansheng Biotechnological Co., LTD., China) were separated into small clumps after 3 min of incubation with TrypLE Express. Then the dissociated cells were transferred to ultralow-attachment plates in E8 basal medium (Gibco, USA). Briefly, H9-ESC colonies (Hangzhou Yuansheng Biotechnological Co., LTD., China) were subjected to a dissociation process using TrypLE Express. After a 3-minute incubation period, the dissociated cells were subsequently transferred to ultralow-attachment plates. Following a period of 6 days, the embryoid bodies (EBs) were collected and placed into culture plates containing MSC induction medium. This medium consisted of 10% fetal bovine serum (FBS, Gibco, USA), high-glucose Dulbecco's modified Eagle's medium (H-DMEM, Gibco, USA), 1 mM L-glutamine (Thermo Fisher, China). After another 14 days, the EB outgrowths were cultured by TrypLE Express. The cells used in this study were hESC-MSCs and labeled as passage 0 (P0). The hESC-MSCs exhibited a homogeneous cell population characterized by a spindle-shaped morphology. Following the completion of five passages, the hESC-MSCs were collected for the purpose of characterization or further expansion to be utilized in further experimental procedures. Cell surface markers and fluorescence-activated cell sorting (FACS) were used to characterize the cells using FACS Calibur flow cytometer with Cell Quest software (BD, Biosciences). The identification of MSCs in this investigation involved the use of several fluorescently tagged monoclonal antibodies for phenotypic staining. hESC-MSCs were characterized with PE/Cy7 antibodies against human CD73; PE antibodies against human CD79A, CD105, CD34, and HLA-DR; FITC antibodies against human CD45; and APC antibodies against human CD90 (all form BD Biosciences, USA). 2.3 Trilineage differentiation assay We investigated the multi differentiation potential of hESC-MSCs toward the osteogenic, adipogenicand chondrogenic lineages in vitro. For osteogenic differentiation, 2 × 10 4 cells evenly distributed and cultured in 24-well plates. The medium was replaced with osteogenic differentiation medium (OriCell™ human mesenchymal stem cell osteogenic differentiation medium, Cyagen Biosciences, China) after the cell confluence reached 60–70%, and then incubated for 3 weeks. Alizarin Red (Cyagen Biosciences) staining was performed to assess the potential of osteogenic differentiation. for adipogenic differentiation, cells were planted at a density of 2×10 4 cells/well on a 24-well plate and cultured in mediums until they approached 100% confluence. Then, the cells were incubated with OriCell™ hMSC adipogenic differentiation medium (Cyagen Biosciences) for a duration of three weeks. The presence of lipid droplets in the differentiated cells was determined by performing Oil Red O staining (Cyagen Biosciences). 4×10 5 cells were cultivated for 4 weeks in OriCell™ hMSC chondrogenic differentiation medium (Cyagen Biosciences) for chondrogenic differentiation.The chondrogenic pellet was collected and preserved in a 4% paraformaldehyde solution after a period of 4 weeks. After embedding and sectioning, chondrogenic differentiation potential was detected by Alcian Blue (Cyagen Biosciences) staining. 2.4 Establishment of the OM animal model and experimental design The OM model was prepared by physical damage and chemical burns as we previously reported 16 . Briefly, after the animal was anesthetized with isoflurane, a 5 mm biopsy punch was used to create a homogeneous standardized circular ulcer on the right cheek of each rat, a soft cotton swab was applied vertically, and the cotton tip was immersed in 50% glacial acetic acid for 30 s. All rats were randomly divided into two groups: the control group (n = 20) and the hESC-MSCs group (n = 20). The OM group was subcutaneously injected with an isolated hESC-MSCs solution (1×10 7 cells suspended in 0.1 ml of PBS) at the four sites on the edge of the ulcer at a depth of 2 to 3 mm by a No. 27 syringe on day 1. The control group was injected with an equal amount of PBS solution. 2.5 Ulcer areas assessment The ulcer areas were assessed on day 0, 2, 4, 7, 10 and 14 after ulcer onset. The animals were promptly administered isoflurane for anesthesia, following which digital cameras (Cannon, Japan) were used to capture images of the oral ulcers. Ulcer areas were measured using ImageJ software 6.0 (Media Cybernetics Inc., Rockville, MD, USA). 2.6 Immunohistochemical and histological assessment The tissue sections were surgically removed and preserved in a solution of 4% paraformaldehyde for a duration of 24 to 48 hours. Subsequently, the tissues were embedded in paraffin, sliced into sections with a thickness of 5 micrometers, and affixed onto slides. For pathological testing, the slides underwent a process of deparaffinization and subsequent staining with PCNA antibody (1:50, rabbit polyclonal, Proteintech, China), H&E (for general examination). Apoptosis in the paraffin-embedded mucosal tissue sections were examined by a TUNEL assay kit (Roche, USA). Three sections were chosen in a random manner for each rat and subjected to staining using the TUNEL assay kit, following the instructions provided by the manufacturer. Three fields of view were selected randomly for each slide were examined. 2.7 Cell culture and treatment HaCaT cells (Chinese Academy of Sciences Cell Bank) stimulated by with LPS (Thermo Fisher, China) or 5-FU (Sigma-Aldrich, USA) are widely used as models of inflammatory and chemotherapy-induced injury in the oral mucosa. First, HaCaT cells (1 × 10 4 cells/well) were seeded in 6-well plates for 12 h. Then, HaCaT cells were induced with 20 µg/ml LPS or 8 µg/ml 5-FU for 24 h to establish an injured epithelial cell model. After 24 h, HaCaT cells were washed 3 times with PBS and then incubated in normal medium (NM) with 3 ml H-DMEM containing 15% FBS and 1% penicillin-streptomycin. 2.8 Establishing a coculture system of HaCaT cells and hESC-MSCs A 6-well Transwell system (2.4 cm diameter, 0.4 µm pore size; Corning, USA) was used to establish an indirect coculture system of HaCaT cells and hESC-MSCs. HaCaT cells were seeded in the lower chambers of the Transwell plates at 5×10 5 cells/well, while hESC-MSCs were seeded in the upper chambers of the Transwell plates at a ratio of 1:1. The cells were incubated at 37°C in 5% CO 2 NM, which was composed of H- DMEM with 15% FBS and 1% penicillin-streptomycin. The cells were randomly divided into six groups: NM (HaCaT cells cultured in NM) group, hESC-MSCs group (HaCaT cell and hESC-MSCs coculture group, cultured in NM), LPS group (HaCaT cells pretreated with LPS), LPS + hESC-MSCs group (coculture group pretreated with LPS), 5-FU group (HaCaT cells pretreated with 5-FU) and 5-FU + hESC-MSCs group (coculture group pretreated with 5-FU). After 24 h, the cells were harvested from the lower chambers and subjected to cell proliferation assays, apoptosis assays, and western blot analysis. 2.9 Cell proliferation and apoptosis assays The cell counting kit-8 assay (HY-K0301, MCE, China) was used to assess the proliferation of HaCaT cells at the indicated time points according to the manufacturer’s protocol. A total of 10 µl of CCK-8 reagent was added to each well and incubated for 4 h at 37°C. The absorbance at 450 nm was measured using a FlexStation® 3 Spectrum Microplate Spectrophotometer (Molecular Devices, USA). The apoptosis Annexin V-FITC/PI detection kit (KGA108, KeyGen Biotechology, China) was employed to evaluate apoptosis in HaCaT cells, following the guidelines provided by the manufacturer. The apoptosis assay involved the collection and staining of cells, which were subsequently analyzed using a CytoFLEX S Flow Cytometer(Beckman Coulter, USA). The resulting data were then analyzed using FlowJo software. 2.10 Western blotting The HaCaT cells in the six groups were harvested for their total protein. On 10% denaturing SDS-PAGE gels, 60 micrograms of total protein were loaded before being transferred to PVDF membranes(0.45 µm, Millipore), which were incubated with anti-GAPDH (1:1000, rabbit polyclonal, Goodhere, China), anti-Bcl-2 (1:1000, 12789-1-AP rabbit polyclonal, Proteintech, China), anti-Bax (1:1000, rabbit monoclonal, Invitrogen, USA), Caspase-3 (1:1000, 19677-1-AP, rabbit polyclonal, Proteintech), anti-PCNA (1:1000, ab92552, rabbit monoclonal, Abcam), PI3K (1:1000, bs-20611R rabbit polyclonal, Bioss, China), P-PI3K (1:1000, ab182651 rabbit polyclonal, Abcam, China), anti-AKT (1:1000, 10176-2-AP rabbit monoclonal, Proteintech), and anti-P-AKT (1:1000, 66444-1-Ig rabbit monoclonal, Proteintech) primary antibodies at 4°C overnight. The blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (Boster, China) for 1 h at room temperature. Images were quantified using the ChemDoc MP Imaging System (Bio-Rad, USA) and analyzed with Image Lab 5.2.1 software (Bio-Rad, USA). 2.11 Wound healing assay Scratch assays were employed to assess the impact of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) on the migration of HaCaT cells. In brief, a total of 5 × 105 HaCaT cells were evenly distributed into each well of 6-well plates and allowed to incubate for a duration of 24 hours. Then, the confluent layer of cells was scratched using a sterile 200 µl pipette tip. Images were caputated at 0, 12, and 24 h. The measurement of the scratched areas was performed via Image-Pro Plus 6.0 software. 2.12 Statistical analysis The statistical significance of the data was assessed using GraphPad software (Synergy Software, USA). The statistical analysis was conducted using GraphPad Prism 6.0 software(GraphPad, USA), with a predetermined threshold for statistical significance set at a level of P < 0.05. 3. RESULTS 3.1. Characterization and identification of hESC-MSCs Primary and passaged hESC-MSCs in culture formed an adherent monolayer and had a fibroblast-like morphology with spindle-shaped cells (Fig. 1 a). The hESC-MSCs exhibited positive expression of the MSC markers CD73, CD90, and CD105, while demonstrating negative expression of the hematopoietic stem cell markers CD14, CD34, CD45, and major histocompatibility protein HLA-DR, as determined through the use of Flow Cytometry (Fig. 1 c). We undertook trilineage differentiation assays to verify the multipotency of hESC-MSCs. hESC-MSCs could differentiate into mesenchymal lineages, as demonstrated by Alcian Blue staining for chondrogenesis, Oil red O staining for adipogenesis, Alizarin Red S staining for osteogenesis (Fig. 1 b). Therefore, hESC-MSCs displayed comparable phenotypic, morphological and functional features to primary MSCs. 3.2. hESC-MSCs promote the healng of OM in the animal model We used a punch with a diameter of 5 mm and further applied 50% glacial acetic acid to create an OM model in rat buccal to assess the reparative effects of hESC-MSCs on mucosal healing. Ulcerations formed 48 h later, which were recorded as day 0. The hESC-MSCs groups were uniformly injected with 100 µl of PBS containing 1×107 hESC-MSCs in the center and around of the ulcer. Ulcerations were employed similarly by PBS in the control group. A whitish-yellow necrotic membrane was present, covering the ulceration, and this manifestation was observed across all groups on day 0. On day 7, the ulcer pseudomembrane in the experimental group disappeared, leaving local swelling red, and the ulcer areas were smaller than those in the NC group, while the ulcers in the control group were still covered the white pseudomembrane. On day 10, the ulcers in the experimental group healed completely, but the ulcers in the NC group were still covered with a thin pseudomembrane. During the experiment, it was observed that the hESC-MSCs group exhibited a significantly reduced ulcer area compared to the control group (P < 0.05), indicating that hESC-MSCs significantly accelerated mucosal healing (Fig. 2 b). We recorded the body weights of rats in the hESC-MSCs group and the control group. Their body weights all decreased on day 2. The body weight in the hESC-MSCs group started to recover from day 4 and that in the control group from day 7 (*P < 0.05) (Fig. 2 c). These results indicated that hESC-MSCs could promote the healing of OM and relieve pain associated with eating. Histopathological manifestations of wound healing in the hESC-MSCs group and the control group at day 2,4,7, and 10 are shown in Fig. 3 d. The histopathologic manifestations of the sections were classified into 4 grades (Table 1 ). HE staining revealed that the pathological scores of the hESC-MSCs group began to be lower than those of the control group on day 4. On day 4, the oral mucosa of rats in the control group had a high level of inflammatory cell infiltration, interstitial edema and extravasation of erythrocytes, with a healing grade was 4; however, the oral mucosa of rats in the hESC-MSCs group had moderate inflammatory cell infiltration, minor interstitial edema, and erythrocyte extravasation, with healing grade was 3. Table 1 Oral wound healing standard by light microsope Grade Pathological manifestations 1 Normal epithelium; no or little cellular infiltration; no hemorrhagic areas; no ulcerations 2 Mild re-epithelialization and vasodilation; mild inflammatory infiltration with mononuclear prevalence; no hemorrhagic areas, edema, ulcerations 3 Moderate epithelial degradation, vasodilation, inflammatory infiltration with neutrophilia; The presence of hemorrhagic areas, edema and eventual ulceration 4 Severe vasodilation and edema, inflammatory infiltration with neutrophils prevalence On day 10, stratified epithelium cells developed, covering the whole ulcer surface of rats in the hESC-MSCs group's oral mucosa. In the deep area of the ulcer, there was no evident inflammatory cell infiltration, and no tiny blood vessels were congested or dilated. The healing score was 1. The control group's oral mucosa had a modest number of inflammatory cells, but there was no evident interstitial edema or erythrocyte extravasation, and the healing grade was 2. To determine the effect of hESC-MSCs on tissue proliferation and epithelialization, immunohistochemistry was performed to detect the expression of PCNA in the control group and hESC-MSCs group on days 2, 4, 7, 10, and 14(Fig. 2 e). The results showed that hESC-MSCs significantly increased the expression of PCNA in hESC-MSCs-injected mucosal tissues compared with that in PBS-injected mucosal tissues throughout the experimental period. The hESC-MSCs group showed higher numbers of PCNA-positive nuclei in the suprabasal and basal cells of the epithelium, as well as in certain migrating keratinocytes at the surface of the ulcer. Additionally, the hESC-MSCs group displayed a higher number of PCNA-positive nuclei in the connective tissue cells compared to the control group. To determine the effect of hESC-MSCs on tissue apoptosis, TUNEL staining was performed on tissue sections in the control group and hESC-MSCs group(Fig. 2 f). At 7 and 14 days, a significant reduction in apoptosis was observed in the hESC-MSCs group compared to the control group, suggesting that hESC-MSCs inhibited chemical- and trauma-induced apoptosis in epithelial cells in vivo. These findings indicated that hESC-MSCs have the ability to promote the proliferation of keratinocytes located in the basal layer and enhance migration. This helps to re-epithelialize wounds and promote OM healing. 3.3. hESC-MSCs inhibit LPS- or 5-FU-injured apoptosis of HaCaT cells and stimulate their proliferation in vitro To investigate the biological effect of hESC-MSCs on apoptosis and proliferation in LPS- or 5-FU-injured epithelial cells, HaCaT cells were treated with 20 µg/ml LPS or 8 µg/ml 5-FU for 24 h and then treated with NM or cocultured with hESC-MSCs (at a ratio of HaCaT cells to hESC-MSCs of 1:1). Flow cytometry analysis was undertook following a 24 h treatment. The apoptosis rates of HaCaT cells cocultured with hESC-MSCs were significantly lower than those in the LPS or 5-FU group, as shown in Fig. 3 a-b. The apoptosis rates in the NM group, hESC-MSCs group, LPS group, LPS + hESC-MSCs group, 5-FU group, and 5-FU + hESC-MSCs group were 2.73 ± 0.072%, 3.25 ± 0.38%, 24.85 ± 0.48%, 7.23 ± 0.12%, 26.92 ± 0.58%, and 10.53 ± 0.64%, respectively. Cellular proliferation was assessed using CCK-8 assays at 24 h. In the LPS + hESC-MSCs and 5-FU + hESC-MSCs groups, the quantity of HaCaT cells were sharply increased compared with those in the LPS or 5-FU group, indicating that hESC-MSCs significantly increased HaCaT cell viability(Fig. 3 c). Furthermore, the western blotting analysis was employed to detect the expression of apoptosis-associated proteins Caspase-3, BCL-2 and Bax in HaCaT cells. The levels of Caspase-3 and Bax in HaCaT cells in the LPS and 5-FU groups were higher than those in the NM group, while the levels were lower than those in the LPS + hESC-MSCs and 5-FU + hESC-MSCs groups. Conversely, the levels of BCL-2 in the LPS group and 5-FU group were lower than those in the NM group, while the levels were higher than those in the LPS + hESC-MSCs and 5-FU + hESC-MSCs groups (Fig. 3 d). Further investigation was conducted to examine the levels of proteins associated with proliferation in the six groups. As shown in Fig. 3 d, the levels of the proliferative -related protein PCNA in HaCaT cells were notably elevated in both the LPS + hESC-MSCs and 5-FU + hESC-MSCs groups compared to the LPS and 5-FU groups. The above findings suggested that hESC-MSCs could inhibit apoptosis in LPS- or 5-FU-injured epithelial cells and promote proliferation. 3.4. hESC-MSCs promote the migration and wound closure of LPS- or 5-FU-injured HaCaT cells The migration of hESC-MSCs was analyzed by wound healing assays. Confluent HaCaT cells were scratched to form a linear wound after being exposed to 20 g/ml LPS or 8 g/ml 5-FU for 24 hours. The HaCaT cells were subsequently cultured with NM or co-cultured with hESC-MSCs (at a ratio of HaCaT cells to hESC-MSCs of 1:1). Marking the wound margin on the image and quantifying the wound healing rate at 12 h and 24 h (Fig. 4 a). At 12 h, wound closure was 23.49 ± 1.31% in the LPS group and 35.71 ± 1.15% in the LPS + hESC-MSCs group. Wound closure was 24.46 ± 2.22% in the 5-FU group and 30.64 ± 0.12% in the 5-FU + hESC-MSCs group(Fig. 4 b). At 24 h, wound closure was 26.34 ± 1.12% in the LPS group and 58.82 ± 1.17% in the LPS + hESC-MSCs group. Wound closure was 27.67 ± 2.18% in the 5-FU group and 34.22 ± 0.38% in the 5-FU + hESC-MSCs group (Fig. 4 c). The results of the wound healing assay showed that hESC-MSCs could significantly promote migration and increase the rate of wound closure by LPS- or 5-FU-injured HaCaT cells compared with NM in vitro. 3.5. hESC-MSCs activate the PI3K/AKT pathway but not the MAPK/ERK pathway in LPS- or 5-FU-injured HaCaT cells Next, we sought to identify the molecular mechanisms involved in hESC-MSCs-mediated inhibition of LPS- or 5-FU-injured apoptosis and promotion of proliferation in HaCaT cell in vivo and in vitro. The PI3K/AKT and MAPK/ERK signaling pathways are essential in cell apoptosis and proliferation 17 , and we considered that PI3K/AKT and MAPK/ERK signaling could potentially play a role in the cellular responses of hESC-MSCs towards LPS- or 5-FU-injured HaCaT cells. The results of western blot analysis demonstrated that phospho-PI3K and phospho-AKT levels in HaCaT cells that were pretreated with LPS or 5-FU were lower than those in the NM group, while those in the LPS + hESC-MSCs group and 5-FU + hESC-MSCs group were increased compared with the NM group. However, there was no difference in the phosphorylation level of ERK in the 5-FU group, FU + hESC-MSCs group, LPS group and LPS + hESC-MSCs group. These results suggested that the PI3K/AKT pathway but not the MAPK/ERK pathway might be necessary for hESC-MSCs-mediated apoptosis inhibition (Fig. 5 a). Then, we examined whether hESC-MSCs acted on HaCaT cells through the PI3K/AKT pathway. We treated the LPS- or 5-FU -injured HaCaT cells in the presence or the absence of LY294002. Our study revealed that the LY294002 treatment reversed the effect of hESC-MSCs' reduction of apoptotic cell numbers(Fig. 5 b). Additionally, western blot analysis showed that hESC-MSCs activated AKT, which increased PCNA and BCL-2 protein levels and decreased Bax and Caspase-3 protein levels, and these effects were reversed by LY294002 (Fig. 5 c-f). These results indicated that hESC-MSCs mediate the PI3K/AKT pathway to modulate apoptosis injured by LPS or 5-FU in HaCaT cells as well as promote their proliferation. 4. DISCUSSION This study investigated the effectiveness and possible mechanisms of hESC-MSCs in OM healing. In this study, we found that hESC-MSC therapy could effectively promote OM healing, and its protective effect might be associated with mediating the PI3K/AKT signaling pathway, thereby reducing LPS or 5-FU-injured apoptosis and promoting cell proliferation. These effects contribute to attenuate the inflammatory cascade response of LPS- or 5-FU-injured HaCaT cells and ultimately facilitate repair activities. The following evidence supports these results: ( 1 ) injection of hESC-MSCs significantly attenuated 50% glacial acetic acid-induced oral mucosal injury in rats; ( 2 ) co-culturing with hESC-MSCs decrease LPS- or 5-FU-injured apoptosis, promoted their proliferation; ( 3 ) the effects of hESC-MSCs on apoptosis inhibition and cell proliferation promotion were reversed by the PI3K inhibitor LY294002. OM is a highly impairing adverse effect of conventional anticancer therapies. Mucositis is observed in roughly 40% of individuals undergoing chemotherapy, with this prevalence rising to nearly 90% among patients with head and neck cancer (HNC) who undergo to combined chemotherapy and radiotherapy treatments. 19% of the latter group will be hospitalized and receive delayed anticancer treatment for high-grade mucositis, leading to an inferior standard of life and a poorer prognosis 1 . Chemoradiotherapy-induced oral mucositis (OM) is a complex biological phenomenon that is marked by amplified inflammation, reduced cell proliferation, accelerated cell senescence/apoptosis, and compromised regenerative capabilities in both the mucosal and submucosal areas 2 . The currently accepted OM pathological process can be divided into five stages that occur sequentially and are mechanistically linked to one another: initiation, the damage response, signaling and amplification, ulceration, and re-epithelialization 18 . There is an increasing number of evidence indicating that MSCs have the potential to alleviate the severity of OM in preclinical animal models. 19,20 . MSCs release a combination of growth factors, cytokines and other substances that are essential to the process of wound healing, which might involve in inducing angiogenesis, reducing inflammation, promoting cell differentiation and migration, collagen formation and restructuring 21 . Therefore, some researchers believe that MSC-based regenerative therapy could be a promising treatment option for chemoradiotherapy-induced OM 2,5,9,22 . hESC-MSCs represent a promising alternative source of cells compared to adult MSCs. These hESC-MSCs offer several distinct advantages, including a notable propensity for rapid proliferation, remarkable potential for differentiation into various cell types, and favorable characteristics associated with anti-inflammatory and immunomodulatory properties. 23,24 . This is, as far as I am aware, the first experimental study to treat OM in rats with hESC-MSCs and investigate the underlying mechanisms. Based on the findings of our study, the hESC-MSCs injection demonstrated a notable enhancement in the healing process of oral mucosa wounds. This was achieved through the acceleration of re-epithelialization, an increase in the production of PCNA, and a reduction in apoptosis, as observed in vivo. We first used a biopsy punch combined with 50% glacial acetic acid to create an oral mucosal ulcer with a uniform size. Then, hESC-MSCs were injected at the time the most severe ulcer occurred (48 h later). The ulcer area was recorded on days 2, 4, 7, and 10 after hESC-MSCs injection. Throughout the duration of the experiment, the hESC-MSCs group consistently had a reduced ulcer area compared to the NM group. Notably, the ulcer completely healed by day 10. Over the experimental course, the hESC-MSCs group exhibited a higher quantity of anti-PCNA-positive nuclei than the NM group. These results are in line with the gradual rise in the proliferation of epithelial cell. The data presented in this study indicate that hESC-MSCs injection enhances the healing process of experimentally produced oral ulcers. However, the possible mechanism for this therapeutic effect has yet to be elucidated. Since LPS has long been known to induce inflammatory mediator production and 5-FU is a standard treatment for chemotherapy-induced mucositis, we established an in vitro model of OM in our study by administering LPS or 5-FU to cultured HaCaT cells. In vitro, we found that hESC-MSCs inhibited LPS- or 5-FU-injured apoptosis in HaCaT cells, enhanced proliferation and migration. We further examined its mechanisms. Many researchers have postulated that the PI3K/AKT signaling pathway assumes a pivotal part in epithelial growth and wound repairing, owing to its recognition as a promising target in cancer treatment and its involvement in diverse cellular processes, such as anabolic metabolism, apoptosis, cell proliferation, and cell migration 25–27 . In our study, we treated LPS- or 5-FU-injured HaCaT cells in the presence and absence of LY294002, which revealed that co-culturing with hESC-MSCs contributed to a reduction in the number of apoptotic cells. Conversely, the effects were reversed by LY294002 treatment(Fig. 5 ). Furthermore, western blot analysis revealed that hESC-MSCs activated AKT, resulting in increased protein levels of PCNA and BCL-2 and decreased protein levels of Bax and Caspase-3, and the PI3K inhibitor LY294002 reversed these results. These findings suggest that hESC-MSCs mediate HaCaT cell apoptosis via activating the PI3K/AKT signaling pathway as well as promote their proliferation. The MAPK/ERK pathway is of significant importance modulating essential cellular activities, like differentiation, migration, proliferation and apoptosis, etc. Dysregulation of this pathway has been associated with cellular transformation and the development of cancer 28,29 . The MAPK/ERK pathway plays a crucial role in promoting cell survival by impeding the activation of pro-apoptotic proteins, such as Bax, while concurrently enhancing the expression of anti-apoptotic proteins, such as BCL-2 30 . There were no differences in ERK phosphorylation levels between the 5-FU and FU + hESC-MSCs groups or between the LPS and LPS + hESC-MSCs groups in our study. These findings suggest that the MAPK/ERK signaling pathway is not required for hESC-MSCs-mediated apoptosis inhibition. This study has several limitations that should not be overlooked. First, this study only referred to relevant literature and used a solitary dosage of hESC-MSCs for the treatment of the animals. In subsequent studies, it will be imperative to ascertain the ideal dosage of hESC-MSCs. Second, there is a lack of knowledge regarding the differentiation of transplanted hESC-MSCs into functional cells for the purpose of tissue regeneration, as well as the possibility of these cells remaining undifferentiated. Further investigation is required in order to comprehensively comprehend the destiny of hESC-MSCs within the mucosal tissue. Third, more research is needed to determine how hESC-MSCs-derived molecules participate in the activation of the PI3K/AKT pathway. Furthermore, future studies should also focus on the long-term systemic effects of hESC-MSC-based therapeutic interventions as well as how the local wound microenvironment affects the injected hESC-MSCs. The findings of our study indicate that there exists a reliable and consistent supply of MSCs that can be utilized for the treatment of OM and other extensive and widespread ulcerations in a clinical setting. Moreover, the results of our study indicate that hESC-MSCs have the ability to suppress LPS- or 5-FU-injured epithelial cell apoptosis, while also promoting cell proliferation through the activation of the PI3K/AKT signaling pathway. The discoveries of this study have significant effects on future research that seek to considering the effectiveness of hESC-MSCs in various functional studies. CONCLUSION Our findings demonstrated that hESC-MSCs injection could effectively promote oral mucosal healing in vivo, inhibit LPS- or 5-FU-injured HaCaT cell apoptosis and promote proliferation in vitro by activating the PI3K/AKT pathway. The results of our study indicate that the hESC-MSCs injection have the potential to serve as an innovative therapeutic approach in the treatment of OM. Declarations Ethics approval and consent to participate The study is reported in accordance with ARRIVE guidelines. All animal experiments were approved by the Institutional Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine authorized the animal research (NO:20201431). Consent for publication Not Applicable. Availability of data and materials All analyzed data are included in the manuscript. The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. Funding This work was supported by the Medical Science and Technology Project of Zhejiang Province (Grant numbers 2021KY684 and 2021PY007). Authors' contributions Kejia Lv: Conceptualization; methodology; software; data curation; investigation; formal analysis; writing – original draft; visualization. Bicong Gao : Methodology; validation. Chenlu Shen: Methodology. Weijia Ye: Supervision. Yanan, Yao: supervision. Hua Yao: Writing – review and editing; funding acquisition; supervision. References Pulito C, Cristaudo A, Porta C, et al. Oral mucositis: the hidden side of cancer therapy. Journal of Experimental & Clinical Cancer Research : CR . Oct 7 2020;39(1):210. doi:10.1186/s13046-020-01715-7 Chen C, Zhang Q, Yu W, Chang B, Le AD. Oral Mucositis: An Update on Innate Immunity and New Interventional Targets. J Dent Res . Sep 2020;99(10):1122-1130. doi:10.1177/0022034520925421 Elad S, Yarom N, Zadik Y, Kuten-Shorrer M, Sonis ST. The broadening scope of oral mucositis and oral ulcerative mucosal toxicities of anticancer therapies. CA: a cancer journal for clinicians . Jan 2022;72(1):57-77. doi:10.3322/caac.21704 Elad S, Cheng KKF, Lalla RV, et al. MASCC/ISOO clinical practice guidelines for the management of mucositis secondary to cancer therapy. Cancer . Oct 1 2020;126(19):4423-4431. doi:10.1002/cncr.33100 Gong W, Wang F, He Y, Zeng X, Zhang D, Chen Q. Mesenchymal Stem Cell Therapy for Oral Inflammatory Diseases: Research Progress and Future Perspectives. Current stem cell research & therapy . 2021;16(2):165-174. doi:10.2174/1574888x15666200726224132 Hendijani F. Human mesenchymal stromal cell therapy for prevention and recovery of chemo/radiotherapy adverse reactions. Cytotherapy . May 2015;17(5):509-25. doi:10.1016/j.jcyt.2014.10.015 Yu HY, Shin JH, Yun H, et al. A Preclinical Study of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells for Treating Detrusor Underactivity by Chronic Bladder Ischemia. Stem cell reviews and reports . Dec 2021;17(6):2139-2152. doi:10.1007/s12015-021-10204-z Shen Z, Wang J, Huang Q, et al. Genetic modification to induce CXCR2 overexpression in mesenchymal stem cells enhances treatment benefits in radiation-induced oral mucositis. Cell death & disease . Feb 14 2018;9(2):229. doi:10.1038/s41419-018-0310-x Maria OM, Shalaby M, Syme A, Eliopoulos N, Muanza T. Adipose mesenchymal stromal cells minimize and repair radiation-induced oral mucositis. Cytotherapy . Sep 2016;18(9):1129-45. doi:10.1016/j.jcyt.2016.06.008 Huang J, U KP, Yang F, et al. Human pluripotent stem cell-derived ectomesenchymal stromal cells promote more robust functional recovery than umbilical cord-derived mesenchymal stromal cells after hypoxic-ischaemic brain damage. Theranostics . 2022;12(1):143-166. doi:10.7150/thno.57234 Lee EJ, Lee HN, Kang HJ, et al. Novel embryoid body-based method to derive mesenchymal stem cells from human embryonic stem cells. Tissue engineering Part A . Feb 2010;16(2):705-15. doi:10.1089/ten.tea.2008.0596 Bahrehbar K, Rezazadeh Valojerdi M, Esfandiari F, Fathi R, Hassani SN, Baharvand H. Human embryonic stem cell-derived mesenchymal stem cells improved premature ovarian failure. World journal of stem cells . Aug 26 2020;12(8):857-878. doi:10.4252/wjsc.v12.i8.857 Xu J, Wang X, Chen J, et al. Embryonic stem cell-derived mesenchymal stem cells promote colon epithelial integrity and regeneration by elevating circulating IGF-1 in colitis mice. Theranostics . 2020;10(26):12204-12222. doi:10.7150/thno.47683 Yoon SY, Yoon JA, Park M, et al. Recovery of ovarian function by human embryonic stem cell-derived mesenchymal stem cells in cisplatin-induced premature ovarian failure in mice. Stem cell research & therapy . Jun 26 2020;11(1):255. doi:10.1186/s13287-020-01769-6 Siddiqi S, Klomjit N, Jiang K, et al. Efficacy of Human Embryonic Stem Cells Compared to Adipose Tissue-Derived Human Mesenchymal Stem/Stromal Cells for Repair of Murine Post-Stenotic Kidneys. Stem cell reviews and reports . Feb 2023;19(2):491-502. doi:10.1007/s12015-022-10443-8 Gao Z, Guan L, Liu Z, et al. Using extracellular vesicles derived from human umbilical cord mesenchymal stem cells for a topical coating promotes oral mucositis healing in rats. Annals of translational medicine . Mar 2022;10(6):290. doi:10.21037/atm-22-767 Jun EK, Zhang Q, Yoon BS, et al. Hypoxic conditioned medium from human amniotic fluid-derived mesenchymal stem cells accelerates skin wound healing through TGF-β/SMAD2 and PI3K/Akt pathways. International journal of molecular sciences . Jan 6 2014;15(1):605-28. doi:10.3390/ijms15010605 Treister N, Sonis S. Mucositis: biology and management. Current opinion in otolaryngology & head and neck surgery . Apr 2007;15(2):123-9. doi:10.1097/MOO.0b013e3280523ad6 Lee DY, Kim HB, Shim IK, Kanai N, Okano T, Kwon SK. Treatment of chemically induced oral ulcer using adipose-derived mesenchymal stem cell sheet. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology . Aug 2017;46(7):520-527. doi:10.1111/jop.12517 Guan Z, Zhang J, Jiang N, Tian M, Wang H, Liang B. Efficacy of mesenchymal stem cell therapy in rodent models of radiation-induced xerostomia and oral mucositis: a systematic review. Stem cell research & therapy . Apr 12 2023;14(1):82. doi:10.1186/s13287-023-03301-y Kraskiewicz H, Paprocka M, Bielawska-Pohl A, et al. Can supernatant from immortalized adipose tissue MSC replace cell therapy? An in vitro study in chronic wounds model. Stem cell research & therapy . Jan 21 2020;11(1):29. doi:10.1186/s13287-020-1558-5 Jung H, Kim HS, Lee JH, Lee JJ, Park HS. Wound Healing Promoting Activity of Tonsil-Derived Stem Cells on 5-Fluorouracil-Induced Oral Mucositis Model. Tissue Eng Regen Med . Feb 2020;17(1):105-119. doi:10.1007/s13770-019-00226-7 Asgari Taei A, Nasoohi S, Hassanzadeh G, Kadivar M, Dargahi L, Farahmandfar M. Enhancement of angiogenesis and neurogenesis by intracerebroventricular injection of secretome from human embryonic stem cell-derived mesenchymal stem cells in ischemic stroke model. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie . Aug 2021;140:111709. doi:10.1016/j.biopha.2021.111709 Yoon D, Yoon D, Sim H, Hwang I, Lee JS, Chun W. Accelerated Wound Healing by Fibroblasts Differentiated from Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells in a Pressure Ulcer Animal Model. Stem cells international . 2018;2018:4789568. doi:10.1155/2018/4789568 Hoxhaj G, Manning BD. The PI3K-AKT network at the interface of oncogenic signalling and cancer metabolism. Nature reviews Cancer . Feb 2020;20(2):74-88. doi:10.1038/s41568-019-0216-7 Han N, Jia L, Su Y, et al. Lactobacillus reuteri extracts promoted wound healing via PI3K/AKT/β-catenin/TGFβ1 pathway. Stem cell research & therapy . Aug 7 2019;10(1):243. doi:10.1186/s13287-019-1324-8 Mi X, Jiao W, Yang Y, Qin Y, Chen ZJ, Zhao S. HGF Secreted by Mesenchymal Stromal Cells Promotes Primordial Follicle Activation by Increasing the Activity of the PI3K-AKT Signaling Pathway. Stem cell reviews and reports . Jun 2022;18(5):1834-1850. doi:10.1007/s12015-022-10335-x Poh PSP, Seeliger C, Unger M, Falldorf K, Balmayor ER, van Griensven M. Osteogenic Effect and Cell Signaling Activation of Extremely Low-Frequency Pulsed Electromagnetic Fields in Adipose-Derived Mesenchymal Stromal Cells. Stem cells international . 2018;2018:5402853. doi:10.1155/2018/5402853 Li C, Xie X, Liu Z, Yang J, Zuo D, Xu S. Neu5Ac Induces Human Dental Pulp Stem Cell Osteo-/Odontoblastic Differentiation by Enhancing MAPK/ERK Pathway Activation. Stem cells international . 2021;2021:5560872. doi:10.1155/2021/5560872 Yue J, López JM. Understanding MAPK Signaling Pathways in Apoptosis. International journal of molecular sciences . Mar 28 2020;21(7)doi:10.3390/ijms21072346 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3449081","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":242504149,"identity":"2e1966b8-628c-421f-b099-c6c18ae0de33","order_by":0,"name":"Kejia Lv","email":"","orcid":"","institution":"Department of Stomatology, First Affiliated Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kejia","middleName":"","lastName":"Lv","suffix":""},{"id":242504150,"identity":"adca0e78-398e-4a03-9149-366937cbae37","order_by":1,"name":"Bicong Gao","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bicong","middleName":"","lastName":"Gao","suffix":""},{"id":242504151,"identity":"67c358ca-e9a5-4409-92ea-d95124b17fe8","order_by":2,"name":"Chenlu Shen","email":"","orcid":"","institution":"Department of Stomatology, First Affiliated Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenlu","middleName":"","lastName":"Shen","suffix":""},{"id":242504152,"identity":"fff09150-1e42-4f88-87d4-10890de11916","order_by":3,"name":"Weijia Ye","email":"","orcid":"","institution":"Department of Stomatology, Lishui Manicipal Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weijia","middleName":"","lastName":"Ye","suffix":""},{"id":242504153,"identity":"4bef340e-dffd-459b-ad18-3498172d2695","order_by":4,"name":"Yanan Yao","email":"","orcid":"","institution":"Department of Stomatology, First Affiliated Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Yao","suffix":""},{"id":242504154,"identity":"eb5d994d-e383-41f1-84fc-27f5c472385f","order_by":5,"name":"Hua Yao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYHACNiC2YWBsAFI8JGhJI13LYQiTKC0Gxw8fe8xTc96eeUYC44O3bQzy5gS1nElLN+Y5dpuZcUYCs+HcNgbDnQ2EtBzIMZPmYbvNBtTCJs3bxpBgcICQlvNvgFr+neMBamH/TZyWG0BbeNsOSIBsYSZKi+SNZ2mSc/uSDRh7HjZLzjknYbiBkBa+88nHJN58s7M3bE8++OFNmY08QVsUYAoMG8CRKUFAPRDIN8AYhNWOglEwCkbBSAUASuo8cYI2YMwAAAAASUVORK5CYII=","orcid":"","institution":"Department of Stomatology, First Affiliated Hospital, Zhejiang University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Yao","suffix":""}],"badges":[],"createdAt":"2023-10-15 15:14:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3449081/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3449081/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":45375121,"identity":"e700a40e-7c83-47a0-9e58-1c5a9992851c","added_by":"auto","created_at":"2023-10-28 17:20:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2127129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of hESC-MSCs.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Representative photomicrograph of adherent hESC-MSCs formed an adherent monolayer and had a fibroblast-like morphology with spindle-shaped cells. \u003cstrong\u003e(b)\u003c/strong\u003e hESC-MSCs could differentiate into mesenchymal lineages, as demonstrated by Alcian Blue staining for chondrogenesis, Oil red O staining for adipogenesis, Alizarin Red S staining for osteogenesis. \u003cstrong\u003e(c) \u003c/strong\u003eThe hESC-MSCs exhibited positive expression of the MSC markers CD73, CD90, and CD105, while demonstrating negative expression of the hematopoietic stem cell markers CD14, CD34, CD45, and major histocompatibility protein HLA-DR, as determined through the use of Flow Cytometry.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3449081/v1/b66bffec6bc984a612f0d552.png"},{"id":45375122,"identity":"1e39d7f5-74b4-46d8-9bdb-66e7bef3c6e6","added_by":"auto","created_at":"2023-10-28 17:20:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8124013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehESC-MSCs injection in the submucosa of the oral ulcerative injury site of Wistar rats accelerated wound closure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Changes of clinical view of buccal ulcer area at days 0, 2, 4, 7, 10, and 14 after hESC-MSCs were injected subcutaneously around the injury site in Wistar rats. Representative images of buccal ulcer area day 0, 2, 4, 7, 10, and 14 after of hESC-MSCs or PBS injection. \u003cstrong\u003e(b)\u003c/strong\u003e A column chart is used to visually depict the average ulcer surface area in mm2 within both the control group and and hESC-MSCs group across various time intervals. \u003cstrong\u003e(c)\u003c/strong\u003e Rats in the control group and hESC-MSCs group were weighed.\u003c/p\u003e\n\u003cp\u003eThe control group exhibited a recovery in body weight commencing on day 7, while the hESC-MSCs group demonstrated a recovery starting on day 4. \u003cstrong\u003e(d)\u003c/strong\u003e Representative photomicrographs of H\u0026amp;E stained sections at day 2, 4, 7, 10, and 14 after injection of PBS or hESC-MSCs. \u003cstrong\u003e(e)\u003c/strong\u003e The quantification of epithelial cell proliferation was assessed through immunohistochemistry utilizing PCNA antibodies in sections obtained from buccal mucosal tissues of both the control and hESC-MSCs groups at various time points including days 2, 4, 7, 10, and 14. \u003cstrong\u003e(f)\u003c/strong\u003e The TUNEL assay was employed to assess the level of apoptosis in sections obtained from buccal mucosal tissues of both the control and hESC-MSCs groups at various time points, specifically on day 2, 4, 7, 10, and 14.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3449081/v1/69701f31dde05009bc6c8297.png"},{"id":45376240,"identity":"2b1defb5-03f6-4388-9145-9da80be37a40","added_by":"auto","created_at":"2023-10-28 17:28:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":978071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehESC-MSCs inhibit LPS- or 5-FU-injured apoptosis of HaCaT cells and stimulate their proliferation in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Examination of apoptosis cells in HaCAT cells in different groups. The apoptosis rate of HaCAT cells in NM group, LPS group, 5-FU group, hESC-MSCs group LPS+hESC-MSCs group, 5-FU+hESC-MSCs group were measured by flow-cytometry analysis with FITC/PI staining after co-cultured with hESC-MSCs for 24h. \u003cstrong\u003e(b)\u003c/strong\u003e the percentage of apoptosis cells were measured, and data were presented as the mean±SEM(n=3). \u003cstrong\u003e(c)\u003c/strong\u003e Cell viability in HaCAT cells for 24 h after treatment in NM group, LPS group, 5-FU group, hESC-MSCs group, LPS+hESC-MSCs group, 5-FU+hESC-MSCs group. \u003cstrong\u003e(d)\u003c/strong\u003e Western blot assay for cleaved caspase-3, BCL-2, Bax, PCNA expression in HaCAT cells of different groups. Significance was measured using a two-way ANOVA. \u003cem\u003e*P\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e***P\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3449081/v1/e39e339e4208a61f1d622d38.png"},{"id":45376241,"identity":"5b472978-8d3c-4ca7-b6c8-3dd9b22eac6f","added_by":"auto","created_at":"2023-10-28 17:28:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2222395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehESC-MSCs promoted the migration of LPS- or 5-FU-injured HaCAT cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Representative images of HaCAT cells scratch assays after 12 and 24h.\u003c/p\u003e\n\u003cp\u003eQuantitative analysis of HaCAT cell migration area at 12 h (b) and 24 h\u003cstrong\u003e(c)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003erespectively.\u003c/p\u003e\n\u003cp\u003eWound-healing assay showed that hESC-MSCs exhibited a significant enhancement in LPS- or 5-FU-induced wound closure compared with the NM group. Significance was measured using a two-way ANOVA. \u003cem\u003e*P \u0026lt; \u003c/em\u003e0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e***P\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3449081/v1/93fe907de4eefaf0d946981d.png"},{"id":45375118,"identity":"f13628c6-474c-45cc-b734-ce8f5b34316b","added_by":"auto","created_at":"2023-10-28 17:20:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1368846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehESC-MSCs inhibited apoptosis and promoted proliferation of LPS- or 5-FU -stimulated HaCAT cells by activating PI3K/AKT signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e P-PI3K, p-AKT, p-ERK2 expression in LPS- or 5-FU-stimulated HaCAT cell were detected by western blot analysis The downregulations of the phosphorylation of the protein were prevented by hESC-MSCs. \u003cstrong\u003e(b)\u003c/strong\u003e Cell viability in HaCAT cells for 24 h after treatment in NM group, LPS group, 5-FU group, hESC-MSCs group, LPS+hESC-MSCs group, 5-FU+hESC-MSCs group, LPS+hESC-MSCs+LY294002 group, 5-FU+hESC-MSCs+LY294002 group.At the same time, PI3K inhibitor LY294002(50 μM) was added to the lower chamber. The cell viability was assessed by CCK-8. The percentage of apoptotic HaCAT cells was quantitatively analyzed as shown in (\u003cstrong\u003ec\u003c/strong\u003e) (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003e(d)\u003c/strong\u003e Western blot assay for cleaved caspase-3, BCL-2, Bax and PCNA in LPS- or 5-FU-stimulated HaCAT cells in the presence or absence of LY294002 (50 μM). \u0026nbsp;\u003cstrong\u003e(e)\u003c/strong\u003e Western blot assay for PI3K, p-PI3K, AKT, p-AKT in LPS- or 5-FU -injured HaCAT cells in the presence or absence of LY294002 (50 μM). Significance was measured using a two-way ANOVA. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3449081/v1/b11f4a1d6cbf361bd41d86eb.png"},{"id":49748558,"identity":"3d19fe30-c95a-4f6d-8108-59323c5ac784","added_by":"auto","created_at":"2024-01-17 11:52:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4378059,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3449081/v1/3be0d4a5-54bb-475a-9e7e-ebad1a153dea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"hESC-derived mesenchymal stem cells Promote Oral Mucositis Healing via the PI3K/AKT Pathway","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eOral mucositis (OM) is among the most pervasive adverse reactions caused by radiotherapy or chemotherapy during cancer treatment\u003csup\u003e1,2\u003c/sup\u003e. Chemo/radiotherapy-induced OM primarily manifests as congestion and erosion in the mouth, which is accompanied by a burning sensation and pain, making it difficult for patients to chew and swallow and substantially impairing nutritional intake and quality of life.\u003c/p\u003e \u003cp\u003eDespite the terrible clinical effects of OM, patients have few methods to prevent or minimize this condition. In addition, the few therapies validated by high levels of evidence are not always applicable to all types of OM, and their effects on tissues are not fully understood, leading to large obstacles for patients receiving cancer treatment\u003csup\u003e3\u003c/sup\u003e. Therefore, more efficacious therapies and preventive guidelines for treating OM are urgently required in the clinic\u003csup\u003e4,5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells (MSCs), a type of stem cell, have the capacity to mitigate tissue damage resulting from inflammation and promote the healing process after injury and have been used to prevent and treat adverse chemo/radiotherapy reactions\u003csup\u003e6\u003c/sup\u003e. Apart from their ability to regenerate, MSCs can migrate to injured tissues and exert paracrine effects such as the recruitment of endogenous progenitor cells and the production of growth factors that promote tissue regeneration\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral studies have demonstrated the efficacy of MSC therapy in the treatment of chemotherapy/radiotherapy-induced mucositis\u003csup\u003e5,8,9\u003c/sup\u003e. However, different biological properties of MSC populations may affect their therapeutic efficacy in this disease. Another challenge is obtaining enough MSCs and culturing them in vitro\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe process of isolating and cultivating human embryonic stem cells (hESCs), which possess the unique ability to undergo unlimited self-renewal and differentiate into various cell types, has introduced novel opportunities for deriving MSCs\u003csup\u003e11\u003c/sup\u003e. hESC-derived MSCs (hESC-MSCs) exhibit enhanced attributes such as a consistent phenotypic, significant proliferative capacity, and consistent immunoregulatory functionality. Consequently, these cells are seen as more appropriate for extensive cultivation compared to MSCs generated from alternative sources. Several studies have recently reported that hESC-MSCs have immunomodulatory and regenerative potential in the treatment of a variety of diseases\u003csup\u003e10,12\u0026ndash;15\u003c/sup\u003e. However, no studies have been conducted to investigate their therapeutic effects on OM and their potential mechanism.\u003c/p\u003e \u003cp\u003eTherefore, we hypothesized that hESC-MSCs play a beneficial role in OM repair. To validate our hypothesis, we compared the healing rate in a rat mucosal injury OM model, with and without the injection of hESC-MSCs. Following that investigated the mechanism by which hESC-MSCs promote repair in a lipopolysaccharide (LPS)- or 5-fluorouracil (5-FU)-induced HaCaT cell injury model. We discovered that hESC-MSCs promoted OM wound healing by stimulating the proliferation of epithelial cells and inhibiting their apoptosis in rat models. Furthermore, hESC-MSCs mediate the PI3K/AKT pathway to modulate apoptosis injured by LPS or 5-FU in HaCaT cells as well as promote their proliferation.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animaland ethical considerations\u003c/h2\u003e \u003cp\u003eMale Wistar rats (8\u0026ndash;10 weeks old and 230\u0026ndash;270 g in weight) were obtained from the Zhejiang Academy of Medical Sciences. The Institutional Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine authorized the animal research(NO:20201431).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 hESC-MSCs obtainment and identification\u003c/h2\u003e \u003cp\u003ehESC-MSCs were purchased from Hangzhou Yuansheng Biotechnological Co., LTD, and cultured with the hESC-MSCs medium (Hangzhou Yuansheng Biotechnological Co., LTD., China). Two steps were used to separate hESC-MSCs from hESCs. Briefly, H9-ESC colonies (Hangzhou Yuansheng Biotechnological Co., LTD., China) were separated into small clumps after 3 min of incubation with TrypLE Express. Then the dissociated cells were transferred to ultralow-attachment plates in E8 basal medium (Gibco, USA).\u003c/p\u003e \u003cp\u003eBriefly, H9-ESC colonies (Hangzhou Yuansheng Biotechnological Co., LTD., China) were subjected to a dissociation process using TrypLE Express. After a 3-minute incubation period, the dissociated cells were subsequently transferred to ultralow-attachment plates. Following a period of 6 days, the embryoid bodies (EBs) were collected and placed into culture plates containing MSC induction medium. This medium consisted of 10% fetal bovine serum (FBS, Gibco, USA), high-glucose Dulbecco's modified Eagle's medium (H-DMEM, Gibco, USA), 1 mM L-glutamine (Thermo Fisher, China). After another 14 days, the EB outgrowths were cultured by TrypLE Express. The cells used in this study were hESC-MSCs and labeled as passage 0 (P0). The hESC-MSCs exhibited a homogeneous cell population characterized by a spindle-shaped morphology. Following the completion of five passages, the hESC-MSCs were collected for the purpose of characterization or further expansion to be utilized in further experimental procedures.\u003c/p\u003e \u003cp\u003eCell surface markers and fluorescence-activated cell sorting (FACS) were used to characterize the cells using FACS Calibur flow cytometer with Cell Quest software (BD, Biosciences). The identification of MSCs in this investigation involved the use of several fluorescently tagged monoclonal antibodies for phenotypic staining. hESC-MSCs were characterized with PE/Cy7 antibodies against human CD73; PE antibodies against human CD79A, CD105, CD34, and HLA-DR; FITC antibodies against human CD45; and APC antibodies against human CD90 (all form BD Biosciences, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Trilineage differentiation assay\u003c/h2\u003e \u003cp\u003eWe investigated the multi differentiation potential of hESC-MSCs toward the osteogenic, adipogenicand chondrogenic lineages in vitro. For osteogenic differentiation, 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells evenly distributed and cultured in 24-well plates. The medium was replaced with osteogenic differentiation medium (OriCell\u0026trade; human mesenchymal stem cell osteogenic differentiation medium, Cyagen Biosciences, China) after the cell confluence reached 60\u0026ndash;70%, and then incubated for 3 weeks. Alizarin Red (Cyagen Biosciences) staining was performed to assess the potential of osteogenic differentiation.\u003c/p\u003e \u003cp\u003efor adipogenic differentiation, cells were planted at a density of 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well on a 24-well plate and cultured in mediums until they approached 100% confluence. Then, the cells were incubated with OriCell\u0026trade; hMSC adipogenic differentiation medium (Cyagen Biosciences) for a duration of three weeks. The presence of lipid droplets in the differentiated cells was determined by performing Oil Red O staining (Cyagen Biosciences). 4\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were cultivated for 4 weeks in OriCell\u0026trade; hMSC chondrogenic differentiation medium (Cyagen Biosciences) for chondrogenic differentiation.The chondrogenic pellet was collected and preserved in a 4% paraformaldehyde solution after a period of 4 weeks. After embedding and sectioning, chondrogenic differentiation potential was detected by Alcian Blue (Cyagen Biosciences) staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Establishment of the OM animal model and experimental design\u003c/h2\u003e \u003cp\u003eThe OM model was prepared by physical damage and chemical burns as we previously reported\u003csup\u003e16\u003c/sup\u003e. Briefly, after the animal was anesthetized with isoflurane, a 5 mm biopsy punch was used to create a homogeneous standardized circular ulcer on the right cheek of each rat, a soft cotton swab was applied vertically, and the cotton tip was immersed in 50% glacial acetic acid for 30 s.\u003c/p\u003e \u003cp\u003eAll rats were randomly divided into two groups: the control group (n\u0026thinsp;=\u0026thinsp;20) and the hESC-MSCs group (n\u0026thinsp;=\u0026thinsp;20).\u003c/p\u003e \u003cp\u003eThe OM group was subcutaneously injected with an isolated hESC-MSCs solution (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells suspended in 0.1 ml of PBS) at the four sites on the edge of the ulcer at a depth of 2 to 3 mm by a No. 27 syringe on day 1. The control group was injected with an equal amount of PBS solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Ulcer areas assessment\u003c/h2\u003e \u003cp\u003eThe ulcer areas were assessed on day 0, 2, 4, 7, 10 and 14 after ulcer onset. The animals were promptly administered isoflurane for anesthesia, following which digital cameras (Cannon, Japan) were used to capture images of the oral ulcers. Ulcer areas were measured using ImageJ software 6.0 (Media Cybernetics Inc., Rockville, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunohistochemical and histological assessment\u003c/h2\u003e \u003cp\u003eThe tissue sections were surgically removed and preserved in a solution of 4% paraformaldehyde for a duration of 24 to 48 hours. Subsequently, the tissues were embedded in paraffin, sliced into sections with a thickness of 5 micrometers, and affixed onto slides. For pathological testing, the slides underwent a process of deparaffinization and subsequent staining with PCNA antibody (1:50, rabbit polyclonal, Proteintech, China), H\u0026amp;E (for general examination).\u003c/p\u003e \u003cp\u003eApoptosis in the paraffin-embedded mucosal tissue sections were examined by a TUNEL assay kit (Roche, USA). Three sections were chosen in a random manner for each rat and subjected to staining using the TUNEL assay kit, following the instructions provided by the manufacturer. Three fields of view were selected randomly for each slide were examined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell culture and treatment\u003c/h2\u003e \u003cp\u003eHaCaT cells (Chinese Academy of Sciences Cell Bank) stimulated by with LPS (Thermo Fisher, China) or 5-FU (Sigma-Aldrich, USA) are widely used as models of inflammatory and chemotherapy-induced injury in the oral mucosa. First, HaCaT cells (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in 6-well plates for 12 h. Then, HaCaT cells were induced with 20 \u0026micro;g/ml LPS or 8 \u0026micro;g/ml 5-FU for 24 h to establish an injured epithelial cell model. After 24 h, HaCaT cells were washed 3 times with PBS and then incubated in normal medium (NM) with 3 ml H-DMEM containing 15% FBS and 1% penicillin-streptomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Establishing a coculture system of HaCaT cells and hESC-MSCs\u003c/h2\u003e \u003cp\u003eA 6-well Transwell system (2.4 cm diameter, 0.4 \u0026micro;m pore size; Corning, USA) was used to establish an indirect coculture system of HaCaT cells and hESC-MSCs. HaCaT cells were seeded in the lower chambers of the Transwell plates at 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well, while hESC-MSCs were seeded in the upper chambers of the Transwell plates at a ratio of 1:1. The cells were incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e NM, which was composed of H- DMEM with 15% FBS and 1% penicillin-streptomycin.\u003c/p\u003e \u003cp\u003eThe cells were randomly divided into six groups: NM (HaCaT cells cultured in NM) group, hESC-MSCs group (HaCaT cell and hESC-MSCs coculture group, cultured in NM), LPS group (HaCaT cells pretreated with LPS), LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs group (coculture group pretreated with LPS), 5-FU group (HaCaT cells pretreated with 5-FU) and 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs group (coculture group pretreated with 5-FU). After 24 h, the cells were harvested from the lower chambers and subjected to cell proliferation assays, apoptosis assays, and western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cell proliferation and apoptosis assays\u003c/h2\u003e \u003cp\u003eThe cell counting kit-8 assay (HY-K0301, MCE, China) was used to assess the proliferation of HaCaT cells at the indicated time points according to the manufacturer\u0026rsquo;s protocol. A total of 10 \u0026micro;l of CCK-8 reagent was added to each well and incubated for 4 h at 37\u0026deg;C. The absorbance at 450 nm was measured using a FlexStation\u0026reg; 3 Spectrum Microplate Spectrophotometer (Molecular Devices, USA). The apoptosis Annexin V-FITC/PI detection kit (KGA108, KeyGen Biotechology, China) was employed to evaluate apoptosis in HaCaT cells, following the guidelines provided by the manufacturer. The apoptosis assay involved the collection and staining of cells, which were subsequently analyzed using a CytoFLEX S Flow Cytometer(Beckman Coulter, USA). The resulting data were then analyzed using FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blotting\u003c/h2\u003e \u003cp\u003eThe HaCaT cells in the six groups were harvested for their total protein. On 10% denaturing SDS-PAGE gels, 60 micrograms of total protein were loaded before being transferred to PVDF membranes(0.45 \u0026micro;m, Millipore), which were incubated with anti-GAPDH (1:1000, rabbit polyclonal, Goodhere, China), anti-Bcl-2 (1:1000, 12789-1-AP rabbit polyclonal, Proteintech, China), anti-Bax (1:1000, rabbit monoclonal, Invitrogen, USA), Caspase-3 (1:1000, 19677-1-AP, rabbit polyclonal, Proteintech), anti-PCNA (1:1000, ab92552, rabbit monoclonal, Abcam), PI3K (1:1000, bs-20611R rabbit polyclonal, Bioss, China), P-PI3K (1:1000, ab182651 rabbit polyclonal, Abcam, China), anti-AKT (1:1000, 10176-2-AP rabbit monoclonal, Proteintech), and anti-P-AKT (1:1000, 66444-1-Ig rabbit monoclonal, Proteintech) primary antibodies at 4\u0026deg;C overnight. The blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (Boster, China) for 1 h at room temperature. Images were quantified using the ChemDoc MP Imaging System (Bio-Rad, USA) and analyzed with Image Lab 5.2.1 software (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Wound healing assay\u003c/h2\u003e \u003cp\u003eScratch assays were employed to assess the impact of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) on the migration of HaCaT cells. In brief, a total of 5 \u0026times; 105 HaCaT cells were evenly distributed into each well of 6-well plates and allowed to incubate for a duration of 24 hours. Then, the confluent layer of cells was scratched using a sterile 200 \u0026micro;l pipette tip. Images were caputated at 0, 12, and 24 h. The measurement of the scratched areas was performed via Image-Pro Plus 6.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical significance of the data was assessed using GraphPad software (Synergy Software, USA). The statistical analysis was conducted using GraphPad Prism 6.0 software(GraphPad, USA), with a predetermined threshold for statistical significance set at a level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization and identification of hESC-MSCs\u003c/h2\u003e \u003cp\u003ePrimary and passaged hESC-MSCs in culture formed an adherent monolayer and had a fibroblast-like morphology with spindle-shaped cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The hESC-MSCs exhibited positive expression of the MSC markers CD73, CD90, and CD105, while demonstrating negative expression of the hematopoietic stem cell markers CD14, CD34, CD45, and major histocompatibility protein HLA-DR, as determined through the use of Flow Cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We undertook trilineage differentiation assays to verify the multipotency of hESC-MSCs. hESC-MSCs could differentiate into mesenchymal lineages, as demonstrated by Alcian Blue staining for chondrogenesis, Oil red O staining for adipogenesis, Alizarin Red S staining for osteogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Therefore, hESC-MSCs displayed comparable phenotypic, morphological and functional features to primary MSCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. hESC-MSCs promote the healng of OM in the animal model\u003c/h2\u003e \u003cp\u003eWe used a punch with a diameter of 5 mm and further applied 50% glacial acetic acid to create an OM model in rat buccal to assess the reparative effects of hESC-MSCs on mucosal healing. Ulcerations formed 48 h later, which were recorded as day 0. The hESC-MSCs groups were uniformly injected with 100 \u0026micro;l of PBS containing 1\u0026times;107 hESC-MSCs in the center and around of the ulcer. Ulcerations were employed similarly by PBS in the control group. A whitish-yellow necrotic membrane was present, covering the ulceration, and this manifestation was observed across all groups on day 0.\u003c/p\u003e \u003cp\u003e On day 7, the ulcer pseudomembrane in the experimental group disappeared, leaving local swelling red, and the ulcer areas were smaller than those in the NC group, while the ulcers in the control group were still covered the white pseudomembrane. On day 10, the ulcers in the experimental group healed completely, but the ulcers in the NC group were still covered with a thin pseudomembrane. During the experiment, it was observed that the hESC-MSCs group exhibited a significantly reduced ulcer area compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that hESC-MSCs significantly accelerated mucosal healing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). We recorded the body weights of rats in the hESC-MSCs group and the control group. Their body weights all decreased on day 2. The body weight in the hESC-MSCs group started to recover from day 4 and that in the control group from day 7 (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). These results indicated that hESC-MSCs could promote the healing of OM and relieve pain associated with eating.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistopathological manifestations of wound healing in the hESC-MSCs group and the control group at day 2,4,7, and 10 are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed. The histopathologic manifestations of the sections were classified into 4 grades (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). HE staining revealed that the pathological scores of the hESC-MSCs group began to be lower than those of the control group on day 4. On day 4, the oral mucosa of rats in the control group had a high level of inflammatory cell infiltration, interstitial edema and extravasation of erythrocytes, with a healing grade was 4; however, the oral mucosa of rats in the hESC-MSCs group had moderate inflammatory cell infiltration, minor interstitial edema, and erythrocyte extravasation, with healing grade was 3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOral wound healing standard by light microsope\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePathological manifestations\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal epithelium; no or little cellular infiltration; no hemorrhagic areas; no ulcerations\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMild re-epithelialization and vasodilation; mild inflammatory infiltration with mononuclear prevalence; no hemorrhagic areas, edema, ulcerations\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eModerate epithelial degradation, vasodilation, inflammatory infiltration with neutrophilia; The presence of hemorrhagic areas, edema and eventual ulceration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSevere vasodilation and edema, inflammatory infiltration with neutrophils prevalence\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOn day 10, stratified epithelium cells developed, covering the whole ulcer surface of rats in the hESC-MSCs group's oral mucosa. In the deep area of the ulcer, there was no evident inflammatory cell infiltration, and no tiny blood vessels were congested or dilated. The healing score was 1. The control group's oral mucosa had a modest number of inflammatory cells, but there was no evident interstitial edema or erythrocyte extravasation, and the healing grade was 2.\u003c/p\u003e \u003cp\u003eTo determine the effect of hESC-MSCs on tissue proliferation and epithelialization, immunohistochemistry was performed to detect the expression of PCNA in the control group and hESC-MSCs group on days 2, 4, 7, 10, and 14(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The results showed that hESC-MSCs significantly increased the expression of PCNA in hESC-MSCs-injected mucosal tissues compared with that in PBS-injected mucosal tissues throughout the experimental period. The hESC-MSCs group showed higher numbers of PCNA-positive nuclei in the suprabasal and basal cells of the epithelium, as well as in certain migrating keratinocytes at the surface of the ulcer. Additionally, the hESC-MSCs group displayed a higher number of PCNA-positive nuclei in the connective tissue cells compared to the control group. To determine the effect of hESC-MSCs on tissue apoptosis, TUNEL staining was performed on tissue sections in the control group and hESC-MSCs group(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). At 7 and 14 days, a significant reduction in apoptosis was observed in the hESC-MSCs group compared to the control group, suggesting that hESC-MSCs inhibited chemical- and trauma-induced apoptosis in epithelial cells in vivo. These findings indicated that hESC-MSCs have the ability to promote the proliferation of keratinocytes located in the basal layer and enhance migration. This helps to re-epithelialize wounds and promote OM healing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. hESC-MSCs inhibit LPS- or 5-FU-injured apoptosis of HaCaT cells and stimulate their proliferation in vitro\u003c/h2\u003e \u003cp\u003eTo investigate the biological effect of hESC-MSCs on apoptosis and proliferation in LPS- or 5-FU-injured epithelial cells, HaCaT cells were treated with 20 \u0026micro;g/ml LPS or 8 \u0026micro;g/ml 5-FU for 24 h and then treated with NM or cocultured with hESC-MSCs (at a ratio of HaCaT cells to hESC-MSCs of 1:1).\u003c/p\u003e \u003cp\u003eFlow cytometry analysis was undertook following a 24 h treatment. The apoptosis rates of HaCaT cells cocultured with hESC-MSCs were significantly lower than those in the LPS or 5-FU group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b. The apoptosis rates in the NM group, hESC-MSCs group, LPS group, LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs group, 5-FU group, and 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs group were 2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.072%, 3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38%, 24.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48%, 7.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12%, 26.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58%, and 10.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64%, respectively.\u003c/p\u003e \u003cp\u003eCellular proliferation was assessed using CCK-8 assays at 24 h. In the LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs and 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs groups, the quantity of HaCaT cells were sharply increased compared with those in the LPS or 5-FU group, indicating that hESC-MSCs significantly increased HaCaT cell viability(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Furthermore, the western blotting analysis was employed to detect the expression of apoptosis-associated proteins Caspase-3, BCL-2 and Bax in HaCaT cells. The levels of Caspase-3 and Bax in HaCaT cells in the LPS and 5-FU groups were higher than those in the NM group, while the levels were lower than those in the LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs and 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs groups. Conversely, the levels of BCL-2 in the LPS group and 5-FU group were lower than those in the NM group, while the levels were higher than those in the LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs and 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Further investigation was conducted to examine the levels of proteins associated with proliferation in the six groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, the levels of the proliferative -related protein PCNA in HaCaT cells were notably elevated in both the LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs and 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs groups compared to the LPS and 5-FU groups. The above findings suggested that hESC-MSCs could inhibit apoptosis in LPS- or 5-FU-injured epithelial cells and promote proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. hESC-MSCs promote the migration and wound closure of LPS- or 5-FU-injured HaCaT cells\u003c/h2\u003e \u003cp\u003eThe migration of hESC-MSCs was analyzed by wound healing assays. Confluent HaCaT cells were scratched to form a linear wound after being exposed to 20 g/ml LPS or 8 g/ml 5-FU for 24 hours. The HaCaT cells were subsequently cultured with NM or co-cultured with hESC-MSCs (at a ratio of HaCaT cells to hESC-MSCs of 1:1). Marking the wound margin on the image and quantifying the wound healing rate at 12 h and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). At 12 h, wound closure was 23.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31% in the LPS group and 35.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15% in the LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs group. Wound closure was 24.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22% in the 5-FU group and 30.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12% in the 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs group(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). At 24 h, wound closure was 26.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12% in the LPS group and 58.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17% in the LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs group. Wound closure was 27.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18% in the 5-FU group and 34.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38% in the 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The results of the wound healing assay showed that hESC-MSCs could significantly promote migration and increase the rate of wound closure by LPS- or 5-FU-injured HaCaT cells compared with NM in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5. hESC-MSCs activate the PI3K/AKT pathway but not the MAPK/ERK pathway in LPS- or 5-FU-injured HaCaT cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we sought to identify the molecular mechanisms involved in hESC-MSCs-mediated inhibition of LPS- or 5-FU-injured apoptosis and promotion of proliferation in HaCaT cell in vivo and in vitro. The PI3K/AKT and MAPK/ERK signaling pathways are essential in cell apoptosis and proliferation\u003csup\u003e17\u003c/sup\u003e, and we considered that PI3K/AKT and MAPK/ERK signaling could potentially play a role in the cellular responses of hESC-MSCs towards LPS- or 5-FU-injured HaCaT cells. The results of western blot analysis demonstrated that phospho-PI3K and phospho-AKT levels in HaCaT cells that were pretreated with LPS or 5-FU were lower than those in the NM group, while those in the LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs group and 5-FU\u0026thinsp;+\u0026thinsp;hESC-MSCs group were increased compared with the NM group. However, there was no difference in the phosphorylation level of ERK in the 5-FU group, FU\u0026thinsp;+\u0026thinsp;hESC-MSCs group, LPS group and LPS\u0026thinsp;+\u0026thinsp;hESC-MSCs group. These results suggested that the PI3K/AKT pathway but not the MAPK/ERK pathway might be necessary for hESC-MSCs-mediated apoptosis inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Then, we examined whether hESC-MSCs acted on HaCaT cells through the PI3K/AKT pathway. We treated the LPS- or 5-FU -injured HaCaT cells in the presence or the absence of LY294002. Our study revealed that the LY294002 treatment reversed the effect of hESC-MSCs' reduction of apoptotic cell numbers(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Additionally, western blot analysis showed that hESC-MSCs activated AKT, which increased PCNA and BCL-2 protein levels and decreased Bax and Caspase-3 protein levels, and these effects were reversed by LY294002 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-f). These results indicated that hESC-MSCs mediate the PI3K/AKT pathway to modulate apoptosis injured by LPS or 5-FU in HaCaT cells as well as promote their proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThis study investigated the effectiveness and possible mechanisms of hESC-MSCs in OM healing. In this study, we found that hESC-MSC therapy could effectively promote OM healing, and its protective effect might be associated with mediating the PI3K/AKT signaling pathway, thereby reducing LPS or 5-FU-injured apoptosis and promoting cell proliferation. These effects contribute to attenuate the inflammatory cascade response of LPS- or 5-FU-injured HaCaT cells and ultimately facilitate repair activities.\u003c/p\u003e \u003cp\u003eThe following evidence supports these results: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) injection of hESC-MSCs significantly attenuated 50% glacial acetic acid-induced oral mucosal injury in rats; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) co-culturing with hESC-MSCs decrease LPS- or 5-FU-injured apoptosis, promoted their proliferation; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) the effects of hESC-MSCs on apoptosis inhibition and cell proliferation promotion were reversed by the PI3K inhibitor LY294002.\u003c/p\u003e \u003cp\u003eOM is a highly impairing adverse effect of conventional anticancer therapies. Mucositis is observed in roughly 40% of individuals undergoing chemotherapy, with this prevalence rising to nearly 90% among patients with head and neck cancer (HNC) who undergo to combined chemotherapy and radiotherapy treatments. 19% of the latter group will be hospitalized and receive delayed anticancer treatment for high-grade mucositis, leading to an inferior standard of life and a poorer prognosis\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChemoradiotherapy-induced oral mucositis (OM) is a complex biological phenomenon that is marked by amplified inflammation, reduced cell proliferation, accelerated cell senescence/apoptosis, and compromised regenerative capabilities in both the mucosal and submucosal areas\u003csup\u003e2\u003c/sup\u003e. The currently accepted OM pathological process can be divided into five stages that occur sequentially and are mechanistically linked to one another: initiation, the damage response, signaling and amplification, ulceration, and re-epithelialization\u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere is an increasing number of evidence indicating that MSCs have the potential to alleviate the severity of OM in preclinical animal models. \u003csup\u003e19,20\u003c/sup\u003e. MSCs release a combination of growth factors, cytokines and other substances that are essential to the process of wound healing, which might involve in inducing angiogenesis, reducing inflammation, promoting cell differentiation and migration, collagen formation and restructuring\u003csup\u003e21\u003c/sup\u003e. Therefore, some researchers believe that MSC-based regenerative therapy could be a promising treatment option for chemoradiotherapy-induced OM\u003csup\u003e2,5,9,22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ehESC-MSCs represent a promising alternative source of cells compared to adult MSCs. These hESC-MSCs offer several distinct advantages, including a notable propensity for rapid proliferation, remarkable potential for differentiation into various cell types, and favorable characteristics associated with anti-inflammatory and immunomodulatory properties.\u003csup\u003e23,24\u003c/sup\u003e. This is, as far as I am aware, the first experimental study to treat OM in rats with hESC-MSCs and investigate the underlying mechanisms. Based on the findings of our study, the hESC-MSCs injection demonstrated a notable enhancement in the healing process of oral mucosa wounds. This was achieved through the acceleration of re-epithelialization, an increase in the production of PCNA, and a reduction in apoptosis, as observed in vivo.\u003c/p\u003e \u003cp\u003eWe first used a biopsy punch combined with 50% glacial acetic acid to create an oral mucosal ulcer with a uniform size. Then, hESC-MSCs were injected at the time the most severe ulcer occurred (48 h later).\u003c/p\u003e \u003cp\u003eThe ulcer area was recorded on days 2, 4, 7, and 10 after hESC-MSCs injection. Throughout the duration of the experiment, the hESC-MSCs group consistently had a reduced ulcer area compared to the NM group. Notably, the ulcer completely healed by day 10.\u003c/p\u003e \u003cp\u003eOver the experimental course, the hESC-MSCs group exhibited a higher quantity of anti-PCNA-positive nuclei than the NM group. These results are in line with the gradual rise in the proliferation of epithelial cell. The data presented in this study indicate that hESC-MSCs injection enhances the healing process of experimentally produced oral ulcers. However, the possible mechanism for this therapeutic effect has yet to be elucidated.\u003c/p\u003e \u003cp\u003eSince LPS has long been known to induce inflammatory mediator production and 5-FU is a standard treatment for chemotherapy-induced mucositis, we established an in vitro model of OM in our study by administering LPS or 5-FU to cultured HaCaT cells. In vitro, we found that hESC-MSCs inhibited LPS- or 5-FU-injured apoptosis in HaCaT cells, enhanced proliferation and migration. We further examined its mechanisms. Many researchers have postulated that the PI3K/AKT signaling pathway assumes a pivotal part in epithelial growth and wound repairing, owing to its recognition as a promising target in cancer treatment and its involvement in diverse cellular processes, such as anabolic metabolism, apoptosis, cell proliferation, and cell migration\u003csup\u003e25–27\u003c/sup\u003e. In our study, we treated LPS- or 5-FU-injured HaCaT cells in the presence and absence of LY294002, which revealed that co-culturing with hESC-MSCs contributed to a reduction in the number of apoptotic cells. Conversely, the effects were reversed by LY294002 treatment(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, western blot analysis revealed that hESC-MSCs activated AKT, resulting in increased protein levels of PCNA and BCL-2 and decreased protein levels of Bax and Caspase-3, and the PI3K inhibitor LY294002 reversed these results. These findings suggest that hESC-MSCs mediate HaCaT cell apoptosis via activating the PI3K/AKT signaling pathway as well as promote their proliferation.\u003c/p\u003e \u003cp\u003eThe MAPK/ERK pathway is of significant importance modulating essential cellular activities, like differentiation, migration, proliferation and apoptosis, etc. Dysregulation of this pathway has been associated with cellular transformation and the development of cancer\u003csup\u003e28,29\u003c/sup\u003e. The MAPK/ERK pathway plays a crucial role in promoting cell survival by impeding the activation of pro-apoptotic proteins, such as Bax, while concurrently enhancing the expression of anti-apoptotic proteins, such as BCL-2\u003csup\u003e30\u003c/sup\u003e. There were no differences in ERK phosphorylation levels between the 5-FU and FU + hESC-MSCs groups or between the LPS and LPS + hESC-MSCs groups in our study. These findings suggest that the MAPK/ERK signaling pathway is not required for hESC-MSCs-mediated apoptosis inhibition.\u003c/p\u003e \u003cp\u003eThis study has several limitations that should not be overlooked. First, this study only referred to relevant literature and used a solitary dosage of hESC-MSCs for the treatment of the animals. In subsequent studies, it will be imperative to ascertain the ideal dosage of hESC-MSCs. Second, there is a lack of knowledge regarding the differentiation of transplanted hESC-MSCs into functional cells for the purpose of tissue regeneration, as well as the possibility of these cells remaining undifferentiated. Further investigation is required in order to comprehensively comprehend the destiny of hESC-MSCs within the mucosal tissue.\u003c/p\u003e \u003cp\u003eThird, more research is needed to determine how hESC-MSCs-derived molecules participate in the activation of the PI3K/AKT pathway. Furthermore, future studies should also focus on the long-term systemic effects of hESC-MSC-based therapeutic interventions as well as how the local wound microenvironment affects the injected hESC-MSCs.\u003c/p\u003e \u003cp\u003eThe findings of our study indicate that there exists a reliable and consistent supply of MSCs that can be utilized for the treatment of OM and other extensive and widespread ulcerations in a clinical setting. Moreover, the results of our study indicate that hESC-MSCs have the ability to suppress LPS- or 5-FU-injured epithelial cell apoptosis, while also promoting cell proliferation through the activation of the PI3K/AKT signaling pathway. The discoveries of this study have significant effects on future research that seek to considering the effectiveness of hESC-MSCs in various functional studies.\u003c/p\u003e "},{"header":"CONCLUSION","content":"\u003cp\u003eOur findings demonstrated that hESC-MSCs injection could effectively promote oral mucosal healing in vivo, inhibit LPS- or 5-FU-injured HaCaT cell apoptosis and promote proliferation in vitro by activating the PI3K/AKT pathway. The results of our study indicate that the hESC-MSCs injection have the potential to serve as an innovative therapeutic approach in the treatment of OM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study is reported in accordance with ARRIVE guidelines. All animal experiments were approved by the Institutional Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine authorized the animal research (NO:20201431).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll analyzed data are included in the manuscript. The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;the\u0026nbsp;Medical Science and Technology Project of Zhejiang\u0026nbsp;Province\u0026nbsp;(Grant numbers\u0026nbsp;2021KY684\u0026nbsp;and 2021PY007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKejia Lv:\u003c/strong\u003e Conceptualization; methodology; software; data curation; investigation; formal analysis; writing \u0026ndash; original draft; visualization. \u003cstrong\u003eBicong Gao\u003c/strong\u003e: Methodology; validation. \u003cstrong\u003eChenlu Shen:\u003c/strong\u003e Methodology. \u003cstrong\u003eWeijia Ye:\u003c/strong\u003e Supervision. \u003cstrong\u003eYanan, Yao:\u003c/strong\u003e supervision. \u003cstrong\u003eHua Yao:\u003c/strong\u003e Writing \u0026ndash; review and editing; funding acquisition; supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePulito C, Cristaudo A, Porta C, et al. Oral mucositis: the hidden side of cancer therapy. \u003cem\u003eJournal of Experimental \u0026amp; Clinical Cancer Research : CR\u003c/em\u003e. Oct 7 2020;39(1):210. doi:10.1186/s13046-020-01715-7\u003c/li\u003e\n\u003cli\u003eChen C, Zhang Q, Yu W, Chang B, Le AD. Oral Mucositis: An Update on Innate Immunity and New Interventional Targets. \u003cem\u003eJ Dent Res\u003c/em\u003e. Sep 2020;99(10):1122-1130. doi:10.1177/0022034520925421\u003c/li\u003e\n\u003cli\u003eElad S, Yarom N, Zadik Y, Kuten-Shorrer M, Sonis ST. The broadening scope of oral mucositis and oral ulcerative mucosal toxicities of anticancer therapies. \u003cem\u003eCA: a cancer journal for clinicians\u003c/em\u003e. Jan 2022;72(1):57-77. doi:10.3322/caac.21704\u003c/li\u003e\n\u003cli\u003eElad S, Cheng KKF, Lalla RV, et al. MASCC/ISOO clinical practice guidelines for the management of mucositis secondary to cancer therapy. \u003cem\u003eCancer\u003c/em\u003e. Oct 1 2020;126(19):4423-4431. doi:10.1002/cncr.33100\u003c/li\u003e\n\u003cli\u003eGong W, Wang F, He Y, Zeng X, Zhang D, Chen Q. Mesenchymal Stem Cell Therapy for Oral Inflammatory Diseases: Research Progress and Future Perspectives. \u003cem\u003eCurrent stem cell research \u0026amp; therapy\u003c/em\u003e. 2021;16(2):165-174. doi:10.2174/1574888x15666200726224132\u003c/li\u003e\n\u003cli\u003eHendijani F. Human mesenchymal stromal cell therapy for prevention and recovery of chemo/radiotherapy adverse reactions. \u003cem\u003eCytotherapy\u003c/em\u003e. May 2015;17(5):509-25. doi:10.1016/j.jcyt.2014.10.015\u003c/li\u003e\n\u003cli\u003eYu HY, Shin JH, Yun H, et al. A Preclinical Study of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells for Treating Detrusor Underactivity by Chronic Bladder Ischemia. \u003cem\u003eStem cell reviews and reports\u003c/em\u003e. Dec 2021;17(6):2139-2152. doi:10.1007/s12015-021-10204-z\u003c/li\u003e\n\u003cli\u003eShen Z, Wang J, Huang Q, et al. Genetic modification to induce CXCR2 overexpression in mesenchymal stem cells enhances treatment benefits in radiation-induced oral mucositis. \u003cem\u003eCell death \u0026amp; disease\u003c/em\u003e. Feb 14 2018;9(2):229. doi:10.1038/s41419-018-0310-x\u003c/li\u003e\n\u003cli\u003eMaria OM, Shalaby M, Syme A, Eliopoulos N, Muanza T. Adipose mesenchymal stromal cells minimize and repair radiation-induced oral mucositis. \u003cem\u003eCytotherapy\u003c/em\u003e. Sep 2016;18(9):1129-45. doi:10.1016/j.jcyt.2016.06.008\u003c/li\u003e\n\u003cli\u003eHuang J, U KP, Yang F, et al. Human pluripotent stem cell-derived ectomesenchymal stromal cells promote more robust functional recovery than umbilical cord-derived mesenchymal stromal cells after hypoxic-ischaemic brain damage. \u003cem\u003eTheranostics\u003c/em\u003e. 2022;12(1):143-166. doi:10.7150/thno.57234\u003c/li\u003e\n\u003cli\u003eLee EJ, Lee HN, Kang HJ, et al. Novel embryoid body-based method to derive mesenchymal stem cells from human embryonic stem cells. \u003cem\u003eTissue engineering Part A\u003c/em\u003e. Feb 2010;16(2):705-15. doi:10.1089/ten.tea.2008.0596\u003c/li\u003e\n\u003cli\u003eBahrehbar K, Rezazadeh Valojerdi M, Esfandiari F, Fathi R, Hassani SN, Baharvand H. Human embryonic stem cell-derived mesenchymal stem cells improved premature ovarian failure. \u003cem\u003eWorld journal of stem cells\u003c/em\u003e. Aug 26 2020;12(8):857-878. doi:10.4252/wjsc.v12.i8.857\u003c/li\u003e\n\u003cli\u003eXu J, Wang X, Chen J, et al. Embryonic stem cell-derived mesenchymal stem cells promote colon epithelial integrity and regeneration by elevating circulating IGF-1 in colitis mice. \u003cem\u003eTheranostics\u003c/em\u003e. 2020;10(26):12204-12222. doi:10.7150/thno.47683\u003c/li\u003e\n\u003cli\u003eYoon SY, Yoon JA, Park M, et al. Recovery of ovarian function by human embryonic stem cell-derived mesenchymal stem cells in cisplatin-induced premature ovarian failure in mice. \u003cem\u003eStem cell research \u0026amp; therapy\u003c/em\u003e. Jun 26 2020;11(1):255. doi:10.1186/s13287-020-01769-6\u003c/li\u003e\n\u003cli\u003eSiddiqi S, Klomjit N, Jiang K, et al. Efficacy of Human Embryonic Stem Cells Compared to Adipose Tissue-Derived Human Mesenchymal Stem/Stromal Cells for Repair of Murine Post-Stenotic Kidneys. \u003cem\u003eStem cell reviews and reports\u003c/em\u003e. Feb 2023;19(2):491-502. doi:10.1007/s12015-022-10443-8\u003c/li\u003e\n\u003cli\u003eGao Z, Guan L, Liu Z, et al. Using extracellular vesicles derived from human umbilical cord mesenchymal stem cells for a topical coating promotes oral mucositis healing in rats. \u003cem\u003eAnnals of translational medicine\u003c/em\u003e. Mar 2022;10(6):290. doi:10.21037/atm-22-767\u003c/li\u003e\n\u003cli\u003eJun EK, Zhang Q, Yoon BS, et al. Hypoxic conditioned medium from human amniotic fluid-derived mesenchymal stem cells accelerates skin wound healing through TGF-\u0026beta;/SMAD2 and PI3K/Akt pathways. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e. Jan 6 2014;15(1):605-28. doi:10.3390/ijms15010605\u003c/li\u003e\n\u003cli\u003eTreister N, Sonis S. Mucositis: biology and management. \u003cem\u003eCurrent opinion in otolaryngology \u0026amp; head and neck surgery\u003c/em\u003e. Apr 2007;15(2):123-9. doi:10.1097/MOO.0b013e3280523ad6\u003c/li\u003e\n\u003cli\u003eLee DY, Kim HB, Shim IK, Kanai N, Okano T, Kwon SK. Treatment of chemically induced oral ulcer using adipose-derived mesenchymal stem cell sheet. \u003cem\u003eJournal of oral pathology \u0026amp; medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology\u003c/em\u003e. Aug 2017;46(7):520-527. doi:10.1111/jop.12517\u003c/li\u003e\n\u003cli\u003eGuan Z, Zhang J, Jiang N, Tian M, Wang H, Liang B. Efficacy of mesenchymal stem cell therapy in rodent models of radiation-induced xerostomia and oral mucositis: a systematic review. \u003cem\u003eStem cell research \u0026amp; therapy\u003c/em\u003e. Apr 12 2023;14(1):82. doi:10.1186/s13287-023-03301-y\u003c/li\u003e\n\u003cli\u003eKraskiewicz H, Paprocka M, Bielawska-Pohl A, et al. Can supernatant from immortalized adipose tissue MSC replace cell therapy? An in vitro study in chronic wounds model. \u003cem\u003eStem cell research \u0026amp; therapy\u003c/em\u003e. Jan 21 2020;11(1):29. doi:10.1186/s13287-020-1558-5\u003c/li\u003e\n\u003cli\u003eJung H, Kim HS, Lee JH, Lee JJ, Park HS. Wound Healing Promoting Activity of Tonsil-Derived Stem Cells on 5-Fluorouracil-Induced Oral Mucositis Model. \u003cem\u003eTissue Eng Regen Med\u003c/em\u003e. Feb 2020;17(1):105-119. doi:10.1007/s13770-019-00226-7\u003c/li\u003e\n\u003cli\u003eAsgari Taei A, Nasoohi S, Hassanzadeh G, Kadivar M, Dargahi L, Farahmandfar M. Enhancement of angiogenesis and neurogenesis by intracerebroventricular injection of secretome from human embryonic stem cell-derived mesenchymal stem cells in ischemic stroke model. \u003cem\u003eBiomedicine \u0026amp; pharmacotherapy = Biomedecine \u0026amp; pharmacotherapie\u003c/em\u003e. Aug 2021;140:111709. doi:10.1016/j.biopha.2021.111709\u003c/li\u003e\n\u003cli\u003eYoon D, Yoon D, Sim H, Hwang I, Lee JS, Chun W. Accelerated Wound Healing by Fibroblasts Differentiated from Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells in a Pressure Ulcer Animal Model. \u003cem\u003eStem cells international\u003c/em\u003e. 2018;2018:4789568. doi:10.1155/2018/4789568\u003c/li\u003e\n\u003cli\u003eHoxhaj G, Manning BD. The PI3K-AKT network at the interface of oncogenic signalling and cancer metabolism. \u003cem\u003eNature reviews Cancer\u003c/em\u003e. Feb 2020;20(2):74-88. doi:10.1038/s41568-019-0216-7\u003c/li\u003e\n\u003cli\u003eHan N, Jia L, Su Y, et al. Lactobacillus reuteri extracts promoted wound healing via PI3K/AKT/\u0026beta;-catenin/TGF\u0026beta;1 pathway. \u003cem\u003eStem cell research \u0026amp; therapy\u003c/em\u003e. Aug 7 2019;10(1):243. doi:10.1186/s13287-019-1324-8\u003c/li\u003e\n\u003cli\u003eMi X, Jiao W, Yang Y, Qin Y, Chen ZJ, Zhao S. HGF Secreted by Mesenchymal Stromal Cells Promotes Primordial Follicle Activation by Increasing the Activity of the PI3K-AKT Signaling Pathway. \u003cem\u003eStem cell reviews and reports\u003c/em\u003e. Jun 2022;18(5):1834-1850. doi:10.1007/s12015-022-10335-x\u003c/li\u003e\n\u003cli\u003ePoh PSP, Seeliger C, Unger M, Falldorf K, Balmayor ER, van Griensven M. Osteogenic Effect and Cell Signaling Activation of Extremely Low-Frequency Pulsed Electromagnetic Fields in Adipose-Derived Mesenchymal Stromal Cells. \u003cem\u003eStem cells international\u003c/em\u003e. 2018;2018:5402853. doi:10.1155/2018/5402853\u003c/li\u003e\n\u003cli\u003eLi C, Xie X, Liu Z, Yang J, Zuo D, Xu S. Neu5Ac Induces Human Dental Pulp Stem Cell Osteo-/Odontoblastic Differentiation by Enhancing MAPK/ERK Pathway Activation. \u003cem\u003eStem cells international\u003c/em\u003e. 2021;2021:5560872. doi:10.1155/2021/5560872\u003c/li\u003e\n\u003cli\u003eYue J, L\u0026oacute;pez JM. Understanding MAPK Signaling Pathways in Apoptosis. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e. Mar 28 2020;21(7)doi:10.3390/ijms21072346\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Oral mucositis, human embryonic stem cell-derived mesenchymal stem cells, Oral mucosal injury, PI3K/AKT signaling, Transwell coculture system","lastPublishedDoi":"10.21203/rs.3.rs-3449081/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3449081/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eOral mucositis (OM) is among the most pervasive adverse reactions caused by radiotherapy or chemotherapy during cancer treatment. This study focused on the reparative effects of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) in OM and possible mechanisms.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eAn ulcer model was created in the rat buccal mucosa to simulate OM, and hESC-MSCs were injected 48h later to assess their reparative effects. The efficacy of hESC-MSCs in regulating apoptosis and proliferation in LPS- or 5-FU-injured HaCaT cells was studied in vitro using a transwell coculture system. Subsequently, the PI3K inhibitor LY24002 was used to assess whether hESC-MSCs regulated injured HaCaT cells through PI3K/AKT pathway.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that hESC-MSCs injection promoted OM healing in rats through the acceleration of re-epithelialization, and a decrease in apoptosis. Our findings also revealed that the hESC-MSCs treatment led to a reduction in the quantity of HaCaT cells undergoing apoptosis. Western blot analysis revealed that hESC-MSCs activated AKT, resulting in increased protein levels of PCNA and BCL-2, decreased protein levels of Bax and Caspase-3. Whereas, LY294002 reversed these changes.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ehESC-MSCs promoted OM healing, inhibited LPS- or 5-FU-injured HaCaT cell apoptosis, and increased their proliferation via the PI3K/AKT pathway.\u003c/p\u003e","manuscriptTitle":"hESC-derived mesenchymal stem cells Promote Oral Mucositis Healing via the PI3K/AKT Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-28 17:20:23","doi":"10.21203/rs.3.rs-3449081/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"198753f6-4498-40df-a7c5-e75bca610e46","owner":[],"postedDate":"October 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-17T11:44:18+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-28 17:20:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3449081","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3449081","identity":"rs-3449081","version":["v1"]},"buildId":"pf3fE39SIOqb-0xH_OWvX","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}