Epidermal stem cells participate in the repair of scalds via Nanog and Myc regulation
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- Published online on: October 24, 2022 https://doi.org/10.3892/mmr.2022.12881
- Article Number: 364
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Copyright: © Yin et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Stem cell therapy has been reported to be effective for injury healing. Growth, wound healing and cell replacement are considered to be a function of stem cells. The mesenchymal stem cells (MSCs) are the most well studied (1) and can differentiate into epidermal cells of skin appendages, including the sebaceous and sweat glands (2). The potential of stem cells to heal skin wounds is evident. However, basic stem cell transplantation does not guarantee successful skin wound healing (3). Therefore, it is necessary to investigate different therapeutic approaches using stem cells in skin wound healing.
Epidermal stem cells (EpSCs) are located at the epidermal base layer and their potential for stem cell production for tissue repair is significant (4,5). Clinical approaches in wound treatment closely rely on EpSCs to maintain skin homeostasis and facilitate wound healing (6). Moreover, enrichment using EpSCs within cultured epidermal autografts helps treat severe wounds (7,8). The biological activity of stem cells can also further improve wound healing (9). For example, Nanog controls the fate of pluripotent inner cell masses during embryonic development via maintaining pluripotent epiblasts and preventing differentiation (10). Furthermore, Nanog, which regulates induced pluripotent stem cell pluripotency and reprogramming, binds to the OCT4 promoter and enhances embryonic stem cell (ESC) self-renewal via mutation (11). Numerous studies have reported that Nanog is a key transcription factor of stem cells (12,13). Our previous study demonstrated that Nanog is inversely related to the differentiation of EpSCs (14). Furthermore, it was also reported that Nanog and the β-catenin/wnt signaling pathway function in EpSCs self-renewal and differentiation (15). Therefore, the aim of the present study was to upregulate Nanog expression and subsequently assess the proliferation of EpSCs. These cells were then used to explore healing in a scalded rat.
Materials and methods
Experimental animals
In total, 15 specific-pathogen-free male Sprague-Dawley (SD) rats (weight, 140–260 g, 6 weeks old) were purchased from Sanxia University (Hubei, China). The rearing environment was relative humidity of 50–60%, and artificial light and dark for 12 h each. The rats were fed in SPF condition for 7 days. All rats were housed at 22–26°C for 21 days. All animal care and experimental procedures were approved by the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (No. 2020122) (Guangzhou, China). All animal experiments were performed in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 2011) (16). All efforts were made to minimize pain and distress of the experimental animals, and all operations comply with the requirements of Guangzhou Medical University on the welfare of laboratory animals, such as operation under anesthesia, euthanasia.
Isolation of mouse EpSCs
Skin tissue was obtained from the back of neonatal SD rats via plastic surgical procedures, washed in PBS and the connective tissue and subcutaneous fat were removed. The skin sample was sterilized with 70% ethanol, rinsed in PBS and minced into 5-mm wide strips using a sharp scalpel. The strips were treated with 0.25% dispase II (Sigma-Aldrich; Merck KGaA) solution at 4°C overnight. The epidermis was mechanically separated from the dermis and incubated in a solution of 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 30 min to dissociate the cells. Enzyme activity was subsequently blocked using DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and the cells were suspended with a pipette. The cell suspension was filtered through a stainless-steel mesh attached to a 60-mm cell culture plate to remove any remaining tissue pieces. Subsequently, the cells were transferred to a 15-ml centrifuge tube and collected using centrifugation for 5 min at 800 g and room temperature (RT). To select stem cells, 1×106 dissociated epidermal cells were plated onto collagen type IV (100 µg/ml; Sigma-Aldrich; Merck KGaA)-coated dishes at room temperature for 10 min. The unattached cells were removed and the rapidly adherent epidermal cells were cultured in keratinocyte serum-free medium supplemented with epidermal growth factor, bovine pituitary extract (all from Gibco; Thermo Fisher Scientific, Inc.) and 0.05 mM CaCl2 (Sigma-Aldrich; Merck KGaA). These cells were cultured at 37°C with 5% CO2 in a humidified incubator for two days before replacing the medium. The medium was changed every other day. The EpSC isolation procedure was regarded as a standard protocol, which was first reported by Liu et al (17) and Jensen et al (18). Rat EpSCs were successfully isolated and identified using CK15, CK19 and β1-integrin with immunofluorescence as previously described (14).
Lentivirus vector construction and EpSCs transduction
The coding sequence of rat Nanog were amplified using PCR from the GV208: Rat Nanog primers (Chengdu Jingming Haorui Biotechnology Co. Ltd.) with AgeI/AgeI overhangs, were used and the fragment was cloned into pTZ58 (Fermentas; Thermo Fisher Scientific, Inc.). The AgeI/AgeI fragment was sub-cloned into pUbi and pEGFP-C1 (Takara Bio USA, Inc.) to generate ubiquitin-Nanog-EGFP encoding plasmids. To produce transduced lentivirus, pBABE-puro plasmids were co-transfected along with helper plasmids into 293T cells (ATCC, CRL-3216) and the medium was harvested at 36 and 72 h. The lentiviral was used 3rd generation system. Quantity of lentiviral plasmid used 5 µg for transfection, and the ratio is 3:2:1 ratio of the lentivirus, packaging and envelope plasmids. The MOI is 10, the interval time is 72 h. The lentivirus vector was construction by OriGene Wuxi Biotechnology Co., Ltd. EpSC transduction was performed by incubating the cells in virus-enriched medium for 12 h, which contained 4 µg/ml polybrene. The transduced EpSCs were divided into a lentivirus-Nanog overexpression (LV-Nanog) group and a control group (LV), which was transduced with the control lentivirus vector (OriGene Wuxi Biotechnology Co., Ltd.) which is empty vector. The Nanog primer sequences are: (accession no. NM001100781) forward, CCGTTGGGCTGACATGAGCGT and reverse, GGCAGGCATCGGCGAGGAAT.
Establishment of EpSCs with stable knockdown of Nanog via transfection of small interfering (si)RNA
The isolated EpSCs were cultured at 37°C in a 12-well plate (5×104 cells/well) for 24 h prior to transfection and the 12-well plate was coated with collagen type IV (100 µg/ml). Transfections were performed using Lipofectamine® RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions for 30 mins at RT. Following transfection, incubated cells for 1–3 days at 37°C. Then, analyze transfected cells and do the subsequent experiments. The following siRNAs were purchased from Qiagen (QIAGEN Shenzhen Company Limited): AllStars Negative Control siRNA (10 µm, cat. no. 1027281); AllStars Hs Cell Death siRNA (10 µm, cat. no. 1027298); Rn_Nanog_2 FlexiTube siRNA (10 µm, cat. no. NM_001100781; GeneGlobe ID, SI02949135). All the siRNA work concentration were kept as 5–10 nM.
Cell proliferation assay
The EpSCs were seeded into 96-well plates, which were coated with collagen type IV, at a density of 2×103 cells/100 µl media/well. The cells were subsequently divided into the following five groups: i) Cells without any treatment; ii) cells transfected with negative control siRNA cells; iii) cells transfected with Nanog-siRNA; iv) cells transduced with LV; and v) cells transduced with LV-Nanog. In brief, 20 µl CellTiter 96® AQueous One Solution Reagent was added to each well (100 µl media/20 µl MTS reagent; cat. no. G3582; Promega Corporation). Subsequently, 20 µl of MTS was added to each well after seeding on day 1–5 of incubation and cultured in the incubator at 37°C. After 1 h of incubation, a microplate reader (Thermo Fisher Scientific, Inc.) was used to assess the absorbance at 490 nm. All experiments were repeated independently three times and measurements from three duplicate wells were used to average each sample.
Wound healing assay
The EpSCs were seeded into six-well plates, which were coated with collagen type IV, at a density of 2×105 cells/1,000 µl media/well. The cells were incubated at 37°C for 24 h to let them adhere to the plate without serum. Scratch wounds were created in confluent cell monolayers using a sterile 200-µl pipette tip and 80% Confluence on either side of the wound at the start of the assay. After 48 h of incubation suspended cells were removed by washing with PBS. Images of the cells were captured immediately using the EVOS Cell Imaging System f1 (Thermo Fisher Scientific, Inc. cat. no: AMF5000). Digitized images of the wounds were analyzed using ImageJ software 3.0 (National Institutes of Health). Wound closure rates were determined as the difference between the wound width at 0 and 48 h.
Western blotting
The control and treated cells were lysed in ice-cold RIPA lysis buffer (Thermo Fisher Scientific, 89900) with protease inhibitors (Thermo Fisher Scientific, 78440) and the protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol. A total of 30 µg protein/lane was separated by 4–12% NuPage Bis-Tirs gels (Thermo Fisher Scientific, NP0336) and then transferred to a PVDF membranes (Bio-Rad, 1620264) on ice (300 mA for 2 h). The membrane was subsequently blocked with 5% non-fat milk dissolved in TBST (10% TBS, 1% tween) buffer with for 1 h at room temperature. Subsequently, the membranes were incubated with the following primary antibodies against: Nanog (1:1,000; cat. no. 3580); cellular (c)-Myc (1:1,000; cat. no. 9402); p53 (1:1,000; cat. no. 9282); and β-actin (1:10,000; cat. no. 5125) (all purchased from Cell Signaling Technology, Inc.) overnight at 4°C. After vigorous washing with TBST (6×5 min), the membranes were subjected to incubation with anti-mouse-IgG-HRP (purchased from Cell Signaling Technology, Inc. 1:1,000; cat. no. 7076) for 1 h at room temperature. The membranes were then washed again with TBST (6×5 min), incubated with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.cat. no. 32209) and imaged using Bio-Rad equipment. Further analysis, as well as image processing and quantification of the bands, was performed using the program Image Lab3.0 (Bio-rad Laboratories, Inc.).
Establishment of the rat scald model and EpSC treatment
The rats were randomly divided into three groups: the normal EpSCs group, the LV-Nanog EpSCs group, the Nanog-siRNA EpSCs group. The rats were intraperitoneally anesthetized using 3% sodium pentobarbital (60 mg/kg) before scalding. All animal procedures conformed to the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (No. 2020122; Guangzhou, China) A self-made constant temperature and constant pressure scald instrument was used to create the model. Immediately following the scald, 5 ml sodium lactate Ringer's solution was injected intraperitoneally for anti-shock. The burn model was produced by the specialized machine under the following conditions: Temperature, 85°C; injury pressure of the scald stick, 0.5 kg; scalding head area, 5 cm2; and scalding time, 8 sec. A stable second-degree scald model was obtained. The area of the scald was 2.25 cm2 (diameter, 1.5 cm). Each rat had one wound on the middle back and bleeding was stopped by appropriate pressure. On the second day of modeling, the test group was injected with 1×105 EpSCs with different levels of Nanog expression using wet compresses, and the control group was injected with PBS using wet compresses. All wounds received pressure dressings. All the group was injected with 200 µl PBS with cells or without cells.
Sample collection and H&E staining
Images of the scalded areas of five rats in each group were captured on days 0, 5, 10, 14 and 21 following injury. The area of each scald was imaged immediately (data not shown). On day 21 following injury, three rats were randomly selected in each group and anesthetized with 3% sodium pentobarbital. 100 mg/kg sodium pentobarbital was used to sacrifice the animal to make all the rats were dead at the end of the experiment. No rats died unexpectedly during our experiments. Death was verified by the absence of a heartbeat and no breathing. For rodents, overdose of sodium pentobarbital is the most preferable euthanizing agent (19,20). Samples from the backs of the rats were received and fixed at room temperature (RT) using 4% polyphosphate formaldehyde (PFA) for 24 h. The samples were embedded in paraffin and sliced into 3-µm thick sections. Slices were dewaxed with xylene for two times (10 min each), hydrated and stained at RT with hematoxylin for 3–6 min, washed with distilled water for 2 min, treated with a mixture of 1% hydrochloric acid in 70% ethanol for 1–3 sec, stained at RT with eosin for 2–3 min, washed with distilled water for 1–2 sec, hydrated with 80% ethanol and 95% ethanol 15–30 sec each and absolute ethanol 1–2 sec, treated with xylene for 2–3 sec twice. The edges of the tissue slices were cleaned using neutral resin and covered with coverslips. Pathological changes were observed using a Leica microscope system with light mode and Leica Application Suite X (Leica Microsystems, Inc.).
Statistical analysis
All the experimental repeats three times and the data was analyzed using PRISM 5.0 software (GraphPad Software, Inc.). Data are presented as the mean ± SEM. Since there was no statistical difference in all data analysis between male and female groups, the data from the male and female groups was pooled. The unpaired Student's t-test was used to determine the statistical differences between the control and experimental groups. One-way ANOVA or multifactorial ANOVA were used for comparisons between multiple groups. A Bonferroni post hoc test was used for pairwise comparisons where appropriate. P<0.05 was considered to indicate a statistically significant difference.
Results
EpSCs proliferative ability is influenced by Nanog expression
The proliferative capability of EpSCs was improved when the cells were transduced with LV-Nanog (Fig. 1). Cell proliferation reached its peak on day 4. Compared with the overexpression of Nanog, the cells treated with Nanog-siRNA exhibited significantly reduced proliferation capabilities (P<0.01). Moreover, both the aforementioned increased proliferation and decreased proliferation were significant compared with the vehicle control group (P<0.05). However, the n.t.-siRNA group and LV control group did not significantly affect cell proliferation.
Western blotting analysis of transduced EpSCs
The successful transduction of LV-Nanog EpSCs was confirmed via western blotting following transduction with LV-Nanog for 4 days. The protein expression levels of Nanog were increased in the LV-Nanog EpSCs compared with the LV-control group (Fig. 2A and C).
Western blotting analysis of Nanog-knockdown EpSCs
EpSCs transfected with siRNAs were subsequently analyzed via western blotting. The results demonstrated that the n.t.-siRNA group did not significantly affect the Nanog protein expression levels. However, in the Nanog-siRNA group Nanog protein expression levels were significantly downregulated (Fig. 2B and D).
Nanog expression affects EpSCs wound healing effectiveness
The wound healing assay was used to detect the effect of Nanog on the healing ability of EpSCs in vitro. The LV-Nanog EpSCs were demonstrated to heal faster compared with the vehicle control, with the wound being almost completely healed after 48 h (Fig. 3A). Moreover, the EpSCs transfected with Nanog-siRNA were demonstrated to heal slower compared with the vehicle control (Fig. 3B).
Nanog promotes EpSCs proliferation via the activation of c-Myc expression
In the aforementioned results it was demonstrated that LV-Nanog EpSCs exhibited increased proliferation. Subsequently, it was demonstrated via western blotting that Nanog protein expression levels were upregulated and the c-Myc protein expression levels were also upregulated in LV-Nanog group (Fig. 4A and B). In the present study, it was demonstrated that when EpSC proliferation is improved via the upregulation of Nanog expression, c-Myc expression is also upregulated. Furthermore, a similar trend was observed in Nanog-siRNA EpSCs, whereby Nanog is downregulated as well as c-Myc (Fig. 4C). The abnormal upregulation of c-Myc expression is directly associated with up to 70% of all human cancers (21,22). The present study demonstrated that c-Myc was upregulated alongside Nanog and potentially improved EpSC proliferation.
The p53 or Arf checkpoints are activated by the persistent expression of oncogenic Myc. Loss of checkpoint regulation via mutations in p53 or Arf demonstrates the full tumorigenic potential of Myc (21). The results of the present study demonstrated that in the LV-Nanog EpSCs c-Myc expression levels were upregulated, along with Nanog expression levels, on day 1 and these peak on day 3. Subsequently, c-Myc expression levels decrease; however, p53 expression levels increase. On day 5, the p53 expression levels were even further upregulated and c-Myc expression levels were decreased compared with those on day 3. Moreover, EpSCs proliferation also reached its peak on day 4 and then decreased. These results suggested that p53 expression was affected by c-Myc expression levels, which potentially may help to avoid c-Myc overexpression. Nanog expression levels in the Nanog-siRNA EpSCs were only slightly upregulated on day 5, which may be due to the efficacy of the siRNA decreasing over time. Moreover, the c-myc expression levels were upregulated on D5 slightly. These results supported the hypothesis that an underlying mechanism of Nanog may be to regulate c-Myc expression to improve the proliferation of EpSCs, a process that may be further supervised by p53.
Scald model of rats treated with EpSCs expressing different levels of Nanog
Once the rat scald model was successfully established, wound healing was investigated on days 0, 5, 10, 14 and 21 (data not shown). The LV-Nanog group healed faster, particularly on day 14 (Fig. 5A). Wound healing was observed in H&E-stained scalded tissue specimen on day 21. The wounds in the control group were the most re-epithelialized. Furthermore, the wounds in the LV-Nanog group were almost completely re-epithelialized on day 21, whereas the wounds in the Nanog-siRNA group were only partially re-epithelialized (Fig. 5B).
Furthermore, the epidermal structure was visible in the normal EpSCs group with only a few inflammatory cells visible on day 21. An intact epidermis was visible in the LV-Nanog group and fresh hair follicle tissues were visible in the dermis.
Discussion
It has previously been demonstrated that EpSCs serve a key role in the wound healing process (4,6,23,24). Furthermore, multipotent EpSCs could enrich from keratinocyte isolates, with their specific location in the hair follicle bulge (5). This result was inconsistent with previous research (15), in this study, the EpSCs proliferation reached its peak on day 4, which was potentially a result of the cell density used and the different of the Nanog expression. Nanog expression maintains EpSC proliferation and the expression of pluripotency genes is maintained by Nanog overexpression (25).
The self-renewal gene, Nanog, is highly expressed in self-renewing embryonic stem cells and has been well studied (12). Nanog is known as one of three ‘core’ factors that allow for stem cell pluripotency (26). Furthermore. Nanog has been reported to also be expressed in several types of adult stem cells, including EpSCs. However, Nanog is nearly not expressed in differentiated cells. In our previous study, it was demonstrated that Nanog served a key role in the regulation of EpSC proliferation (14,15). Stem cell marker expression, such as that of Nanog and Sox-2, is gradually lost as stem cells age (27). Furthermore, our previous study also reported that Nanog expression is reduced in differentiated cells (14). In the present study, it was demonstrated that high Nanog expression levels improved EpSC proliferation and migration. Previous studies have also suggested that in all Nanog target promoters, genes are equally expressed or repressed, whereas in Nanog unique targets genes were predominantly inactivated or suppressed in response to the expression of a subset of target genes. This observation is common to all factors except Myc (28). In an extended transcriptional network for the pluripotency of ESCs it was demonstrated that Nanog and Myc have a large overlap of target promoters; however, Myc also has its own distinct cluster (28). Myc promotes cell proliferation, as previously demonstrated (29–32). Moreover, dominant negative mutants of Myc antagonizes self-renewal and promotes cell differentiation (29). These results suggested that other transcription factors may be involved in this process.
Myc is a transcription factor that regulates cell proliferation (33). Moreover, Myc is widely expressed during embryogenesis, but also in highly proliferative adult tissues, such as the epidermis and gut (34). Myc is sufficient to stimulate proliferation in quiescent cells via overexpression (35). Furthermore, Myc is known as a co-regulator of cell proliferation and metabolism in numerous types of stem cells (28,36). Therefore, the expression of Myc is finely regulated by transcriptional, post-transcriptional and post-translational regulatory mechanisms in adult tissue homeostasis (37,38). Myc is usually maintained at low levels or restricted to regeneration and cell proliferation, such as in the epidermis and the gut. In mouse ES cells, Myc is required to prevent MAPK activation forcing differentiation of cells into primitive endoderm and serves a role in cell proliferation (30). Together, these aforementioned studies demonstrated that c-Myc functionally upregulates energy production and biosynthetic processes required for successful cellular replication, thereby directly coordinating proliferative metabolism and cell cycle progression. In the present study, the results demonstrated that c-Myc protein expression levels were upregulated when Nanog was overexpressed, which potentially increased EpSC proliferation.
Nanog and Myc, along with Krüppel-like factor 4, are naïve pluripotent markers that drive the transition of EpSCs to ESCs (39). Moreover, Nanog and Myc gene expression are independent of each other in the transcriptional regulatory pathways and do not directly regulate each other, but the Nanog and Myc both stimulate cell proliferation (28). Our previous hypothesis that Nanog overexpression, with high c-Myc expression levels, increased EpSC proliferation, was confirmed by the results of the present study. The result was inconsistent with previous research (12). In this study, the EpSCs reach proliferation peak earlier, which was potentially a result of the cell density used.
Furthermore, in the present study it was demonstrated that Nanog overexpression in EpSCs could potentially improve cell proliferation via enhancing c-Myc expression. However, there is also a protection mechanism to prevent excessive proliferation and tumor development (40).
p53 is a well-known critical tumor suppressor and transcription factor (40,41). In the present study, the results demonstrated that c-Myc protein expression was induced when Nanog was overexpressed in EpSCs, which led to EpSC proliferation. However, overexpression of c-Myc protein can induce hepatocellular carcinoma and other types of tumor (42,43). Co-occupancy regions and cis-overlapping motifs of p53 and c-Myc proteins suggest that these two transcription factors interact and regulate gene expression via competitive binding (44). A previous study also demonstrated that intact p53 suppresses Nanog function (45).
Wound healing is a complex process. Self-repair following an injury occurs underneath the skin and tissue where pluripotent adult stem cells have the ability to self-renew and give rise to different cells types (46). Stem cells give rise to progenitor cells, which cannot self-renew but can give rise to numerous cell types. The extent to which stem cells are involved in skin wound healing is complex and not fully understood (3,46). If epithelium formation in the injured area is rapid, healing will lead to regeneration, or scarring will develop over weeks or months. Therefore, epithelization is the one of the most important factors in wound healing and EpSCs are responsible for the process of epidermalization. The rate of epidermalization also depends on the cell proliferation (47). In the present study, according to the rat scald model, it was demonstrated that following treatment with EpSCs being injected into the wound, the wounds in the LV-Nanog group were re-epithelialized by day 21, which was faster compared with the control group. This led to regeneration that did not result in a scar.
Consequently, to better understand the Nanog-stimulated wound healing, the wound healing assay was used with transfected EpSCs. The results demonstrated that Nanog overexpression in EpSCs increased the wound repair capabilities, whereas in EpSCs with Nanog knockdown wound closure was delayed. Different levels of Nanog in the EpSCs may therefore affect the wound closure rate of the scratch. These results suggested that Nanog potential provides a proliferative advantage to EpSCs and that Nanog may be very active in inducing and supporting the wound healing process.
In the present study, the effectiveness of Nanog on EpSCs during the scald healing process was demonstrated. The data suggested that Nanog overexpression in EpSCs could potentially increase the cell proliferation and enhance the wound closure rate. In vivo experiments also supported the hypothesis. When EpSCs expressing LV-Nanog were injected into the rat scald model, the skin re-epithelialized earlier compared with the control group.
In conclusion, in the present study the role of Nanog in EpSC proliferation and migration was investigated. The results demonstrated that in EpSCs, Nanog potentially stimulates cell proliferation and cells that overexpress Nanog may be able to improve wound healing in the rat scald model. Therefore, modifications to EpSC proliferation potential may be an effective way of speeding up the healing of scalds. The present study also demonstrated that the function of Nanog in EpSCs may also be influenced by c-Myc upregulation and p53 control. These data have therefore proposed that this approach could be an effective treatment option to repair wounds to a high quality.
Acknowledgements
Not applicable.
Funding
The present study was supported in part by the Third Affiliated Hospital of Guangzhou Medical University and the Guangdong Medical Science and Technology Research Fund (grant no. B2016026).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.
Authors' contributions
DLY performed data curation, software and formal analysis, validation, investigation, provided methodology, acquired funding, wrote the original draft, wrote reviewed and edited the manuscript. XHZ, QYJ, SL and YL performed data curation, software, formal analysis, validation, investigation and provided methodology. PC conducted software and formal analysis, investigation, wrote, reviewed and edited the manuscript. GQJ conceptualized and supervised the study, provided resources, performed data curation, validation, project administration, wrote, reviewed and edited the manuscript. CYL conceptualized and supervised the study, provided resources, project administration, wrote, reviewed and edited the manuscript. All authors read and approved the final manuscript. DLY and GQJ confirm the authenticity of all the raw data.
Ethics approval and consent to participate
All animal care and experimental procedures in the present study were approved by the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (No. 2020122) (Guangzhou, China).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
Wei X, Yang X, Han ZP, Qu FF, Shao L and Shi YF: Mesenchymal stem cells: A new trend for cell therapy. Acta Pharmacol Sin. 34:747–754. 2013. View Article : Google Scholar : PubMed/NCBI | |
Jeschke MG, Rehou S, McCann MR and Shahrokhi S: Allogeneic mesenchymal stem cells for treatment of severe burn injury. Stem Cell Res Ther. 10:3372019. View Article : Google Scholar : PubMed/NCBI | |
Kosaric N, Kiwanuka H and Gurtner GC: Stem cell therapies for wound healing. Expert Opin Biol Ther. 19:575–585. 2019. View Article : Google Scholar : PubMed/NCBI | |
Yang R, Liu F, Wang J, Chen X, Xie J and Xiong K: Epidermal stem cells in wound healing and their clinical applications. Stem Cell Res Ther. 10:2292019. View Article : Google Scholar : PubMed/NCBI | |
Morasso MI and Tomic-Canic M: Epidermal stem cells: The cradle of epidermal determination, differentiation and wound healing. Biol Cell. 97:173–183. 2005. View Article : Google Scholar : PubMed/NCBI | |
Li Y, Zhang J, Yue J, Gou X and Wu X: Epidermal stem cells in skin wound healing. Adv Wound Care (New Rochelle). 6:297–307. 2017. View Article : Google Scholar : PubMed/NCBI | |
Brockmann I, Ehrenpfordt J, Sturmheit T, Brandenburger M, Kruse C, Zille M, Rose D and Boltze J: Skin-derived stem cells for wound treatment using cultured epidermal autografts: Clinical applications and challenges. Stem Cells Int. 2018:46236152018. View Article : Google Scholar : PubMed/NCBI | |
Teng M, Huang Y and Zhang H: Application of stems cells in wound healing-an update. Wound Repair Regen. 22:151–160. 2014. View Article : Google Scholar : PubMed/NCBI | |
Dash BC, Xu Z, Lin L, Koo A, Ndon S, Berthiaume F, Dardik A and Hsia H: Stem cells and engineered scaffolds for regenerative wound healing. Bioengineering (Basel). 5:232018. View Article : Google Scholar : PubMed/NCBI | |
Silva J, Chambers I, Pollard S and Smith A: Nanog promotes transfer of pluripotency after cell fusion. Nature. 441:997–1001. 2006. View Article : Google Scholar : PubMed/NCBI | |
Hayashi Y, Caboni L, Das D, Yumoto F, Clayton T, Deller MC, Nguyen P, Farr CL, Chiu HJ, Miller MD, et al: Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells. Proc Natl Acad Sci USA. 112:4666–4671. 2015. View Article : Google Scholar : PubMed/NCBI | |
Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M and Yamanaka S: The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 113:631–642. 2003. View Article : Google Scholar : PubMed/NCBI | |
Pashaiasl M, Khodadadi K, Kayvanjoo AH, Pashaei-Asl R, Ebrahimie E and Ebrahimi M: Unravelling evolution of Nanog, the key transcription factor involved in self-renewal of undifferentiated embryonic stem cells, by pattern recognition in nucleotide and tandem repeats characteristics. Gene. 578:194–204. 2016. View Article : Google Scholar : PubMed/NCBI | |
Yin D, Tian L, Ye Y, Li K, Wang J, Cheng P, Chen A, Guo F and Huang H: Nanog and β-catenin: A new convergence point in EpSC proliferation and differentiation. Int J Mol Med. 29:587–592. 2012. View Article : Google Scholar : PubMed/NCBI | |
Cheng P, Sun X, Yin D, Xu F, Yang K, Qin L, Dong Y, Guo F, Chen A, Zhang W and Huang H: Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation. Cell Biosci. 5:52015. View Article : Google Scholar : PubMed/NCBI | |
National Research Council, . Guide for the care and use of laboratory animals. 8th edition. The National Academies Press; Washington, DC: pp. 2462011 | |
Liu Y, Zhou H and Gao F: Isolation and identification of stem cells from adult cashmere goat skin. Int J Dermatol. 47:551–556. 2008. View Article : Google Scholar : PubMed/NCBI | |
Jensen KB, Driskell RR and Watt FM: Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nat Protoc. 5:898–911. 2010. View Article : Google Scholar : PubMed/NCBI | |
Wong D, Makowska IJ and Weary DM: Rat aversion to isoflurane versus carbon dioxide. Biol Lett. 9:201210002012. View Article : Google Scholar : PubMed/NCBI | |
Hickman DL and Johnson SW: Evaluation of the aesthetics of physical methods of euthanasia of anesthetized rats. J Am Assoc Lab Anim Sci. 50:695–701. 2011.PubMed/NCBI | |
Dang CV: MYC on the path to cancer. Cell. 149:22–35. 2012. View Article : Google Scholar : PubMed/NCBI | |
Gabay M, Li Y and Felsher DW: MYC activation is a hallmark of cancer initiation and maintenance. Cold Spring Harb Perspect Med. 4:a0142412014. View Article : Google Scholar : PubMed/NCBI | |
Li L, Gu W, Du J, Reid B, Deng X, Liu Z, Zong Z, Wang H, Yao B, Yang C, et al: Electric fields guide migration of epidermal stem cells and promote skin wound healing. Wound Repair Regen. 20:840–851. 2012. View Article : Google Scholar : PubMed/NCBI | |
Yang R, Wang J, Chen X, Shi Y and Xie J: Epidermal stem cells in wound healing and regeneration. Stem Cells Int. 2020:91483102020. View Article : Google Scholar : PubMed/NCBI | |
Finley LWS, Vardhana SA, Carey BW, Alonso-Curbelo D, Koche R, Chen Y, Wen D, King B, Radler MR, Rafii S, et al: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity. Nat Cell Biol. 20:565–574. 2018. View Article : Google Scholar : PubMed/NCBI | |
Pan G and Thomson JA: Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 17:42–49. 2007. View Article : Google Scholar : PubMed/NCBI | |
Bijonowski BM, Yuan X, Jeske R, Li Y and Grant SC: Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells. Sci Rep. 10:204482020. View Article : Google Scholar : PubMed/NCBI | |
Kim J, Chu J, Shen X, Wang J and Orkin SH: An extended transcriptional network for pluripotency of embryonic stem cells. Cell. 132:1049–1061. 2008. View Article : Google Scholar : PubMed/NCBI | |
Cartwright P, McLean C, Sheppard A, Rivett D, Jones K and Dalton S: LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development. 132:885–896. 2005. View Article : Google Scholar : PubMed/NCBI | |
Wernig M, Meissner A, Cassady JP and Jaenisch R: c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell. 2:10–12. 2008. View Article : Google Scholar : PubMed/NCBI | |
Fagnocchi L, Cherubini A, Hatsuda H, Fasciani A, Mazzoleni S, Poli V, Berno V, Rossi RL, Reinbold R, Endele M, et al: A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity. Nat Commun. 7:119032016. View Article : Google Scholar : PubMed/NCBI | |
Carroll PA, Freie BW, Mathsyaraja H and Eisenman RN: The MYC transcription factor network: Balancing metabolism, proliferation and oncogenesis. Front Med. 12:412–425. 2018. View Article : Google Scholar : PubMed/NCBI | |
Maoz M, Devir M, Inbar M, Inbar-Daniel Z, Sherill-Rofe D, Bloch I, Meir K, Edelman D, Azzam S, Nechushtan H, et al: Clinical implications of sub-grouping HER2 positive tumors by amplicon structure and co-amplified genes. Sci Rep. 9:187952019. View Article : Google Scholar : PubMed/NCBI | |
Spencer CA and Groudine M: Control of c-myc regulation in normal and neoplastic cells. Adv Cancer Res. 56:1–48. 1991. View Article : Google Scholar : PubMed/NCBI | |
Eilers M, Schirm S and Bishop JM: The MYC protein activates transcription of the alpha-prothymosin gene. EMBO J. 10:133–141. 1991. View Article : Google Scholar : PubMed/NCBI | |
Yoon K, Lim YS, Yu SB, Kim DS, Ryu SJ, Kim KH, Jang TH and Kim SH: The expression of survivin and its related genes in adipocyte-derived stem cell by demethylation. Korean J Anesthesiol. 58:383–390. 2010. View Article : Google Scholar : PubMed/NCBI | |
Levens D: You don't muck with MYC. Genes Cancer. 1:547–554. 2010. View Article : Google Scholar : PubMed/NCBI | |
Farrell AS and Sears RC: MYC degradation. Cold Spring Harb Perspect Med. 4:a0143652014. View Article : Google Scholar : PubMed/NCBI | |
Swaidan NT, Salloum-Asfar S, Palangi F, Errafii K, Soliman NH, Aboughalia AT, Wali AHS, Abdulla SA and Emara MM: Identification of potential transcription factors that enhance human iPSC generation. Sci Rep. 10:219502020. View Article : Google Scholar : PubMed/NCBI | |
Pitolli C, Wang Y, Candi E, Shi Y, Melino G and Amelio I: p53-mediated tumor suppression: DNA-damage response and alternative mechanisms. Cancers (Basel). 11:19832019. View Article : Google Scholar : PubMed/NCBI | |
Parfenyev S, Singh A, Fedorova O, Daks A, Kulshreshtha R and Barlev NA: Interplay between p53 and non-coding RNAs in the regulation of EMT in breast cancer. Cell Death Dis. 12:172021. View Article : Google Scholar : PubMed/NCBI | |
Yang P, Jiang Y, Pan Y, Ding X, Rhea P, Ding J, Hawke DH, Felsher D, Narla G, Lu Z and Lee RT: Mistletoe extract Fraxini inhibits the proliferation of liver cancer by down-regulating c-Myc expression. Sci Rep. 9:64282019. View Article : Google Scholar : PubMed/NCBI | |
Nakagawa R, Toboso-Navasa A, Schips M, Young G, Bhaw-Rosun L, Llorian-Sopena M, Chakravarty P, Sesay AK, Kassiotis G, Meyer-Hermann M and Calado DP: Permissive selection followed by affinity-based proliferation of GC light zone B cells dictates cell fate and ensures clonal breadth. Proc Natl Acad Sci USA. 118:e20164251182021. View Article : Google Scholar : PubMed/NCBI | |
Kin K, Chen X, Gonzalez-Garay M and Fakhouri WD: The effect of non-coding DNA variations on P53 and cMYC competitive inhibition at cis-overlapping motifs. Hum Mol Genet. 25:1517–1527. 2016. View Article : Google Scholar : PubMed/NCBI | |
Brandner S: Nanog, Gli, and p53: A new network of stemness in development and cancer. EMBO J. 29:2475–2476. 2010. View Article : Google Scholar : PubMed/NCBI | |
Nourian Dehkordi A, Mirahmadi Babaheydari F, Chehelgerdi M and Raeisi Dehkordi S: Skin tissue engineering: Wound healing based on stem-cell-based therapeutic strategies. Stem Cell Res Ther. 10:1112019. View Article : Google Scholar : PubMed/NCBI | |
Pastar I, Stojadinovic O, Yin NC, Ramirez H, Nusbaum AG, Sawaya A, Patel SB, Khalid L, Isseroff RR and Tomic-Canic M: Epithelialization in wound healing: A comprehensive review. Adv Wound Care (New Rochelle). 3:445–464. 2014. View Article : Google Scholar : PubMed/NCBI |