Umbilical cord subtotipotent-like stem cells reverse the ageing of thymic epithelial cells by inducing reprogramming of the whole-genome methylation and transcription profile

doi:10.21203/rs.3.rs-2374641/v1

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Umbilical cord subtotipotent-like stem cells reverse the ageing of thymic epithelial cells by inducing reprogramming of the whole-genome methylation and transcription profile

https://doi.org/10.21203/rs.3.rs-2374641/v1

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Background: A decrease in the number and activity of thymic epithelial cells (TECs) is an important factor in thymic degeneration, which can lead to dysfunction of T-cell differentiation, development and selection and a decrease in human immune function. Umbilical cord pluripotent-like stem cells (UCSSCs) can promote the structural and functional regeneration of the ageing thymus in vivo, but the mechanism of their action on TECs and molecules is not clear. Based on the discovery that UCSSCs promote thymus regeneration, we further analysed the changes in genome methylation modification profiles and their association with transcription profiles in ageing TECs cocultured with UCSSCs.

Results: UCSSCs could promote thymus regeneration in ageing macaque monkeys, and an ageing model of TECs was established. Coculture of ageing TECs with UCSSCs for 48 hours significantly reduced the expression levels of ageing markers such as P16, P21 and p53 and increased the activity of ageing TECs. Genome-wide methylation and transcriptional sequencing analysis of TECs showed that methylation levels of 501 genes increased and 591 genes decreased in the promoter region. Twenty-three genes with increased methylation were enriched in the processes of negative regulation of cell growth, proliferation and apoptosis, and 37 genes with decreased methylation were enriched in the processes of promoting cell growth and proliferation and inhibiting apoptosis. Association analysis between promoter and transcription level revealed that there was a significant negative correlation between promoter methylation level and gene transcription in 66 genes, among which PDE5A, DUOX2, LAMP1 and SVIL, which inhibit growth and development, showed increased methylation and decreased transcription. Decreased methylation of POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3, which promote cell growth, leads to an increase in transcription levels. Decreased promoter methylation of the transcription factor NGF gene, which leads to an increase in KRT17 and FOXJ1 transcription, plays a key role in reversing TEC senescence.

Conclusion: UCSSC coculture ameliorates the age-associated hallmarks of ageing TECs. The methylation profile of senescent TEC genes was significantly changed after treatment with umbilical cord pluripotent stem cells. It was found that decreasing the methylation level of the transcription factor NGF and increasing the expression level of KRT17 and FOXJ1 play a key role in promoting the proliferation of TECs.

DNA methylation

TECs

UCMSC

Ageing

RNA-seq

The thymus is the main lymphoid organ and is composed of thymic epithelial cells (TECs), dendritic cells and endothelial cells[1, 2]. TECs are composed of cortical TECs (cTECs) and medullary TECs (mTECs). They maintain T-cell development at different stages through single-positive and double-positive selection, so TECs are an indispensable component of T lymphocyte maturation in the thymus[3, 4]. However, in humans and mice, the thymus begins to degenerate after birth, and the reticular structure of the thymus epithelium is gradually replaced by adipose tissue, accompanied by a decrease in T-cell output[5, 6]. Some experiments have shown that TECs have lower proliferation potential and that there is a higher percentage of apoptotic and senescent TECs in elderly animals, indicating that these cells may be a potential target for thymus rejuvenation therapy[7–9]. Therefore, the decrease in the number and activity of TECs may be the key factors related to the unfavourable changes in thymus structure and function in ageing, and the promotion of TEC growth may be the basis by which subtotipotent stem cells promote ageing thymus regeneration.

The best way to reverse thymus ageing is to stimulate growth of the thymus, but this is a challenging scientific and technical problem involving developmental biology. Umbilical cord subtotipotent-like stem cells (UCSSCs) are adult stem cells isolated from neonatal umbilical cord tissue. They are characterized by small size, strong growth ability and high expression of marker molecules of embryonic stem cells (ESCs) and umbilical cord mesenchymal stem cells (UC- MSCs), such as OCT-4, Nanog, SSEA[10, 11] and CD29, CD44, CD105[12, 13]. The differentiation ability is much stronger than that of UC-MSCs, and the effect on tissue damage, degeneration repair and regeneration is stronger[14–16]. In a study on the treatment of ovarian senescence in macaque monkeys, it was found that UCSSCs could reverse the degeneration of ovarian structure and function caused by ageing, by mechanisms such as promoting follicular and vascular regeneration, increasing sex hormone secretion, reducing the degree of ovarian fibrosis and inhibiting apoptosis[17–19]. The evolution of UCSSCs after entering the ageing body, the interaction with the tissue microenvironment and the mechanism of regulating gene modification and expression are not very clear.

DNA methylation is an epigenetic modification mode that regulates gene transcription without changing gene structure. It can turn gene transcription on or off through demethylation or methylation modification of specific genes and their promoters[20, 21]. Previous studies have found that the high stability of DNA methylation is an important mechanism for stabilizing gene transcription, which regulates cell growth, division, proliferation, differentiation and secretory function by regulating gene opening and closing[22, 23]. The methylation level of the functional gene itself is also closely related to the transcriptional expression of the gene, but whether it inhibits or promotes gene transcription is also related to the methylation site. To elucidate the molecular regulatory mechanism by which UCSSCs promote thymus regeneration, we cocultured senescent TECs with UCSSCs to analyse the changes in the genomic methylation and transcriptional profiles to clarify the molecular regulatory mechanism and provide theoretical support for the use of UCSSCs in the treatment of thymus senescence.

After UCSSC treatment in elderly macaque monkeys, UCSSCs were found to promote thymus tissue regeneration by acting on TECs (Fig. 2). To explore the molecular mechanism by which UCSSCs promote thymus regeneration, we further cocultured TECs and UCSSCs in vitro and found that coculture could reverse the ageing phenotype (Fig. 3) of TECs in terms of morphology, structure and function. Whole gene methylation and transcriptome sequencing analysis of senescent and cocultured TECs showed that UCSSCs reversed TEC senescence (Fig. 4) by regulating the level of gene methylation modification. Comparative analysis and association analysis of the database were carried out by using bioinformatics analysis technology, and the changes in methylation and transcriptional profiles in the gene promoter region and gene body region and their correlation (Fig. 5) were revealed. Key regulatory genes were found, and functional verification (Fig. 6) was carried out. This provides a strategy for the treatment of thymic ageing, which is rarely reported at home and abroad (Fig. 1).

Changes in the tissue structure and function of the thymus after UCSSC treatment in elderly macaque monkeys

Elderly macaque monkeys aged 22 to 24 years old were treated with UCSSCs at a dose of 1 × 10~7 cells/kg intravenously once a day for 3 consecutive days. The gross anatomy, morphology, tissue structure and function of the thymus were observed 5 months after the last treatment. The results showed that the thymus tissue of elderly macaque monkeys obviously atrophied and degenerated and was replaced by a large amount of adipose tissue; the boundary between skin and medulla was blurred, and the number of thymocytes decreased. After UCSSC treatment, the thymus tissue regenerated, showing that the volume of the thymus increased, the number of thymus lobules increased, the number of thymus epithelial cells and thymocytes increased, the number of Hassall bodies and the amount of adipose tissue decreased and the thymus index increased (P < 0.05) (Fig. 2A, 2B). The levels of thymosin α and thymopoietin in peripheral blood increased (P < 0.05) (Figure 2C). The expression level of the ageing marker gene P16 increased (Fig. 2D), and the proportion of CK14+ cells expressing TEC marker molecules increased[24, 25] (Fig. 2E). The results showed that UCSSC treatment could improve the structure and function of the thymus and promote the regeneration of thymic lobules.

Intervention effect of UCSSCs on senescent TECs in vitro

To demonstrate that UCSSCs promote thymus tissue regeneration by promoting the growth and differentiation of TECs, we induced the senescence of TECs with 200 μmol/L H₂O₂ in vitro and cocultured UCSSCs with ageing TECs for 48 h with Transwells . β-Gal staining showed that most of the cells in the model group were stained blue, and the number of blue cells decreased significantly after coculture (Fig. 3A). Dynamic observation by a Nanolive 4D microscope showed that UCSSCs secreted vesicles into the ageing TECs during coculture, which changed the volume of the TECs from large to small, and the loose nucleus became compact (Supplementary video 1). Under transmission electron microscopy, heterochromatin aggregation and patchy distribution were observed in senescent TECs, mitochondria were swollen, and no obvious autophagosomes were found in the cells. After coculture, the chromatin distribution of TECs was uniform, the mitochondria were still swollen, and a small number of autophagosomes could be seen in the cells (Fig. 3B). The above results showed that the expression of the ageing-related genes P16 and P21 increased in the model group (Fig. 3C), and UCSSCs cocultured with ageing TECs could reverse TEC senescence; decrease the expression level of ageing marker molecules; improve growth activity, autophagy and viability; and promote TECs to enter the stage of replication and proliferation.

Changes in the genomic methylation profile in senescent TECs after UCSSC coculture

To explore the intervention effect of UCSSCs on the whole-genome methylation modification spectrum of senescent TECs, we used whole-genome methylation sequencing (WGBS) to analyse the whole-genome methylation modification map of TECs after ageing and UCSSC coculture (Fig. 4A). The bioinformatics analysis method was used to conduct a series analysis of the WGBS database, resulting in 76.1 billion WGBS paired terminal readings (Table 2). The average alignment rate of clean WGBS reads mapped to the reference genome (BSMAP (version: 2.90) was 90.8%, and the coverage rate of the reference C base was 95%. We found that the average genome-wide methylation level of TECs in the model group was 64.13%. After coculture, the average methylation level of TECs increased (Fig. 4B). In the site methylation level analysis, 57.58% of the CG sites in the model group were hypermethylated (methylation levels ranged from 70 to 100%). The proportion of hypermethylated CG sites increased after UCSSC coculture, while the proportion of hypomethylated CG sites (methylation level < 30%) decreased after UCSSC coculture (Fig. 4C and Supplementary Fig. 2). Subsequently, we analysed the methylation level around the gene body, and the methylation level decreased gradually in the upstream 1 kb region of the gene. A significant decrease in methylation level was observed closer to the gene transcription initiation site (TSS). The methylation level in the gene body region was higher than that in the adjacent region and was relatively stable, while the methylation level in the transcription termination site (TTS) decreased significantly (Fig. 4D). Furthermore, the methylation changes in the TEC gene regulatory series after UCSSC coculture were determined, and the annotations of senescence-related genes were extracted from the Gene Ontology Consortium (http://wiki.geneontology.org/index.php)). It was found that 1092 genes had methylation differences in the promoter region (Table 3). Among them, the methylation level in the promoter region of 501 genes was increased, and 23 genes were mainly involved in negative regulation of growth, proliferation and promotion of apoptosis. The methylation level of 591 genes decreased in the promoter region; 37 of these genes promoted cell growth, proliferation and anti-apoptosis (Fig. 4E, 4F) (Table 4). These results suggest that UCSSCs methylate the promoter region of genes related to apoptosis promotion, cell cycle arrest and growth inhibition in senescent TECs and turn off the expression of genes related to growth promotion, cell proliferation and apoptosis inhibition.

UCSSCs regulate gene transcription by inducing DNA methylation modification

To study the relationship between the DNA methylation modification pattern and gene transcription during UCSSC reversal of TEC senescence, the genes in TECs were divided into four groups according to transcription level (high, medium, low and negative expression). We found that there was a negative correlation between DNA methylation and transcription in the promoter region (upstream 1 kb), a negative correlation between methylation and transcription in the gene body region near the transcriptional initiation site, and a positive correlation between methylation and transcription as far away from the transcriptional initiation site (Fig. 5A and Supplementary Fig. 3). In addition, the Pearson correlation coefficients (r) between DNA methylation levels and corresponding gene expression levels in each group were calculated. The results showed that DNA methylation levels in promoter and gene body regions were negatively correlated and positively correlated with their transcriptional levels, respectively (Supplementary fig. 4A and B).

After UCSSC coculture, 66 genes in senescent TECs showed a significant negative correlation between promoter methylation and transcriptional level (Table 5). Methylation of PDE5A, DUOX2, LAMP1 and SVIL in the promoter region led to the downregulation of transcription. The expression of these genes mainly inhibited cell development, differentiation and the cell cycle (Fig. 5B and 5C). The demethylation of POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 in the promoter region led to an increase in transcriptional levels, and their expression mainly promotes the cell cycle, cell growth and development and epithelial cell differentiation (Fig. 5B and 5C). KEGG analysis showed that the genes with decreased methylation levels were mainly enriched in the Rap1 signalling pathway (Fig. 5D). Furthermore, the expression of key proteins RAP1, SRC and ITGB3 in the RAP1 signalling pathway was detected by Western blotting. The results showed that the expression of the key proteins RAP1, SRC and ITGB3 increased significantly after UCSSC coculture, suggesting that the decreased methylation of PGF and NGF activated the RAP1 signalling pathway (Fig. 5E). The results showed that UCSSCs induced a decrease in the methylation level in the promoter region of ageing-promoting genes, promoted the demethylation of cell growth gene promoters, inhibited the expression of ageing-promoting genes, promoted an increase in cell growth gene expression, and finally reversed the senescence of TECs. The increased expression level of genes related to the RAP1 signalling pathway played an important role in this process.

In the gene body region, there was a significant negative correlation between methylation level and gene transcription in UCSSC-cocultured senescent TECs, and these genes were defined as BNGs (Table 6) (Fig. 5F). These genes are mainly involved in biological processes such as cell population proliferation, epithelial cell differentiation and regulation of epithelial cell proliferation (Fig. 5G). However, there was a significant positive correlation between the methylation level of more genes (553 risk-related genes) and gene transcription, and these genes were defined as BPGs (Fig. 5F). Interestingly, BPGs also had biological processes similar to those of BNGs (Fig. 5G). These results suggest that methylation changes in the gene body region may not only inhibit gene transcription but may also promote gene transcription in the process by which UCSSCs reverse TEC ageing.

UCSSCs promote the demethylation of the transcription factor NGF and then promote the expression of KRT17 and FOXJ1

Transcription factor binding sequences rich in 66 genes whose methylation levels were negatively related to transcription in the promoter region were identified (Table 7). Based on gene expression correlation and sequence analysis, we established a network map of UCSSCs to reverse the 408 connections between five ageing transcription factors, SVIL, POLR3G, NGF, and TRIM39, and 401 target genes in TEC ageing-related genes (Fig. 6A). In this network, NGF is the central regulatory factor, targeting 108 genes, including many antiaging-related motifs, such as CHTF18, TRIM39, KRT17, PGF, FOXJ1, KNTC1, F2R and POLR3G. Among them, KRT17 and FOXJ1 are targets of NGF, indicating that promoter demethylation is closely related to the increase in mRNA expression during UCSSC reversal of TEC ageing (Pearson r =-0.61 and -0.74, Fig. 6B). In this process, the expression level of NGF was positively correlated with that of KRT17 and FOXJ1 (Pearson's r = 0.92 and 0.74, Fig. 6C). Immunofluorescence analysis and RT‒qPCR further proved that NGF interacted with KRT17 and FOXJ1 (Fig. 6D and E). Functionally, NGF, KRT17 and FOXJ1 were mainly involved in the proliferation of epithelial cells. KI67 showed that the proliferation ability of ageing TECs increased after coculture with UCSSCs (Fig. 6F). In summary, these results suggest that UCSSCs can demethylate the NGF gene of ageing TECs and promote the expression of KRT17 and FOXJ1, thus promoting the growth, mitosis and proliferation of ageing TECs (Fig. 6G).

In this study, we used elderly rhesus monkeys as a model to observe the effect of UCSSCs on thymus regeneration in elderly rhesus monkeys[26]. After UCSSC treatment, thymus volume and mass increased, thymus epithelial cell lines exhibited increased proliferation, the cortex-medulla structure was obviously improved, and TEC marker CK14 expression increased[27–29], indicating that UCSSC treatment promoted thymus regeneration in elderly rhesus monkeys. H₂O₂ is a small molecule oxidant that can induce cell senescence due to oxidative stress caused by high levels of free radicals produced by the cell membrane. It can increase the expression of β-galactosidase (β-Gal) and increase the expression of senescence marker genes P16 and P21[30–32]. After treatment with 200 µmol/L H₂O₂ for 72 h, the expression of β-Gal in TECs was increased. The senescent cells were stained blue by β-galactosidase, and the expression levels of P16 and P21 were increased, which agreed with the characteristics of senescent cells. When UCSSCs were cocultured with senescent TECs in Transwell chambers for 48 h, the expression of β-Gal and senescence-related genes P16 and P21 decreased. Under a Nanolive 4D microscope, it was observed that UCSSCs continuously secreted vesicular substances into ageing TECs during coculture, resulting in uniform distribution of TEC chromatin, appearance of intracellular autophagosomes and smaller cell morphology. Transmission electron microscopy showed that the chromatin concentration of ageing TECs was patchy, and the mitochondria were moderately swollen. After coculture, chromatin was evenly distributed, and autophagosomes were observed[33, 34]. These results suggest that UCSSCs can reverse the ageing TECs, repair TEC damage and restore their growth activity to a certain extent.

Based on the analysis of the results of genome-wide methylation sequencing, it was found that the sites of methylation modification were mainly concentrated in CG loci, which was consistent with reports in mammals[35]. We analysed the methylation level of different positions of the gene and observed significant hypomethylation around the transcriptional start site; the methylation level of the gene body was higher than that of the adjacent region, and the methylation level decreased significantly after the transcriptional end site, indicating that the methylation level changed differently at different locations of the gene[36]. The results of differential methylation gene analysis between groups showed that after UCSSC coculture, 23 genes that inhibited cell growth and proliferation were methylated, and 37 genes that promoted cell growth and anti-inflammatory and antioxidant stress were demethylated to reverse TEC senescence, which indicated that methylation in the promoter region is one of the key mechanisms by which UCSSCs reverse TEC senescence.

It has been confirmed that DNA methylation in the gene promoter region can lead to gene silencing[37]. Our study revealed that the methylation of the ageing-promoting genes PDE5A, DUOX2, LAMP1 and SVIL in the promoter region leads to a decrease in the transcription level, while a decrease in the methylation level of the growth-promoting genes POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 leads to an increase in the transcription level. The Rap1 signalling pathway plays an important role in cell adhesion and integrin function in many cell types[38]. After coculture with UCSSCs, it activates the Rap1 signalling pathway, increases the expression of Rap1, SRC and ITGB3, promotes cell proliferation and facilitates resistance to apoptosis[39–41]. In the gene body region, although some studies have confirmed the effect of gene body DNA methylation on gene expression, the function of gene body DNA methylation is still poorly understood [42, 43]. In our study, UCSSCs in the gene body region not only inhibited gene transcription but also promoted the transcription of some genes in the process of reversing TEC ageing.

DNA methylation in the promoter region prevents binding to transcription factors, leading to gene silencing[44]. Our research shows that NGF gene methylation inhibits the expression of KRT17 and FOXJ1, which highlights the regulatory mechanism of DNA methylation in the process of UCSSC coculture with senescent TECs. Nerve growth factor NGF is reported to exist mainly in medullary epithelial cells of normal and regenerated thymic Hassall bodies, stimulating the activity and proliferation of mouse TECs in vitro[45, 46]. Therefore, NGF may be a candidate regulatory gene in the process of UCSSC coculture of ageing TECs. In addition, KRT17 and FOXJ1, as downstream regulators of NGF, are involved in epithelial cell development, but no studies describing the effects of these genes on TEC senescence have been published, so they may be new markers of TEC cell adhesion, cell proliferation and tissue development.

UCSSC treatment can improve the structure and function of the thymus and promote the regeneration of thymic lobules. Coculture of UCSSCs and ageing TECs in vitro significantly reduced the expression level of ageing marker molecules, promoted the activity of ageing TECs, and reversed the ageing of TECs in terms of morphology, structure and function. The mechanism by which UCSSCs promote thymus regeneration is that they inhibit the expression of senescence genes by inducing methylation in the promoter region of ageing genes, promote the expression of cell growth and differentiation-related genes by inhibiting promoter methylation, and finally reverse TEC senescence. UCSSCs promote the expression of KRT17 and FOXJ1 by increasing the expression of genes related to the RAP1 signalling pathway in ageing TECs and regulating the demethylation of NGF genes. These changes play a key role in reversing TEC senescence, indicating that NGF, KRT17 and FOXJ1 are new markers of senescence.

Animal experiment

Six macaque monkeys were obtained from Kunming Institute of Zoology, Chinese Academy of Sciences [SCXK (Yunnan) K2017-0003]. The macaque monkeys were aged 22-26 years old and were randomly divided into two groups: the senescence model group (n=3) and the senescence treatment group (n=3). P4 generation UCSSCs were diluted with 0.9% sterile sodium chloride solution at a concentration of 2 × 106 cells/ml. After the macaque monkeys in the treatment group were immobilized, UCSSCs were injected into the femoral vein at 1 × 107/kg per monkey once every other day 3 times. The macaque monkeys in the elderly model group were given the same amount of 0.9% normal saline. Experimental animal certificate number: SYXK (military) 2017-0051. Ethical approval for the use of animal models was provided by the Experimental Animal Committee of the 920th Hospital of the Joint Logistics Support Force.

Thymus index measurement

After three months of treatment, macaque monkeys were anaesthetized with more than 3% pentobarbital sodium and euthanized. Then, the following steps were followed: the chest cavity was opened, the heart was located, then the thymus was identified along the great blood vessels, removed, and weighed (g) with an electronic balance; finally, the thymus organ index of rhesus monkeys after treatment was determined.

Thymus tissue HE staining

The thymus tissue was fixed in 4% paraformaldehyde solution for 24 hours and then dehydrated and embedded in paraffin. The thickness of the sections was approximately 4μm. After being treated with xylene-anhydrous ethanol-75% ethanol, the sections were sealed with neutral glue. Sections were observed and photographed with a Nikon microscope (NikonDS-U3, Nikon, Japan).

Determination of thymosin α and thymopoietin levels

After three months of treatment, the serum was isolated from the peripheral blood of macaque monkeys, and the expression levels of thymosin α and thymopoietin in serum were measured by ELISA kits (Millipore).

Detection of P16 expression by immunofluorescence staining

The thymus tissue was fixed in 4% paraformaldehyde solution for 24 hours and then dehydrated and embedded in paraffin. The thickness of the section was approximately 4μm. An anti-rabbit anti-monkey antibody P16 was added for staining. FITC goat anti-rabbit was incubated at room temperature for 50 minutes, and the nucleus was restained with DAPI for 15 minutes. Finally, the sections were sealed with anti-fluorescence quenching sealant and photographed under a microscope.

Immunohistochemical staining of the thymic epithelial cell-specific marker molecule CK14

Thymus tissue section with a thickness of 4 μm were dewaxed in water for antigen repair and blocking of endogenous peroxidase. One anti-mouse anti-monkey CK14 antibody was used, and the second antibody was labelled with HRP. After incubation with the secondary antibody, the nuclei were stained with DAPI. Sections were observed and photographed with a Nikon microscope (Nikon DS-U3, Nikon, Japan). The positive area signal was analysed by ImageJ software (NIH, Bethesda, MD).

TECs

TECs were purchased from Hangzhou Meisen Co. and highly expressed the epithelial cell-specific marker EPCAM. They were cultured in a medium (DMEM/F12) containing 10% calf serum (Invitrogen).

Establishment and identification of the TEC ageing model

Molecular analysis of ageing markers

β-Galactosidase (SA- β-Gal) staining TECs were collected, washed with PBS once, operated according to the kit instructions (Solarbio, G1580), and observed under an ordinary optical microscope (ZEN2011), and the number of blue-positive cells in three visual fields was counted.

Expression level analysis of P21 and P16 TECs were collected for Western blotting analysis and lysed in RIPA buffer (Solarbio, China, Beijing). The total proteins of TEC cells were separated by Tris-Gly and 10% gel electrophoresis and then transferred to a nitrocellulose membrane. The cell membrane was sealed with 5% skim milk at room temperature for 1.5 hours and then incubated with an antibody overnight. The next day, the membrane was washed with PBST and incubated for 30 min with horseradish peroxidase-labelled secondary antibody. Protein bands were detected after treatment with SuperSignal West Femto (Thermo Fisher Science, Waltham, MA, USA). The primary anti-rabbit anti-human P16 (1RV 1000, ab32503), P21 (1RV 1000, ab32351), SRC (1RV 300000 ab179473 ab109381), and ITGB3 (1RV 1000 ab179473) antibodies were obtained from Abcam. The primary anti-rabbit anti-human β-actin antibody (1RV 25000~66009-1-Ig) and the secondary anti-HRP-Goat IgG antibody (1RV 1000, SA00001-2) were purchased from Proteintech.

Observation of the cell substructure by fluoroscopic electron microscopy

TECs were collected, digested with 0.25% trypsin and made into a cell suspension. After washing with PBS, the cell precipitate was centrifuged and fixed for 4 hours. After immersion in acetone, ultrathin sections of 80 nm were stained with uranium and lead, observed under a transmission electron microscope (Hitachi, HT7700), and photographed for image analysis.

Establishment and identification of a coculture model of UCSSCs and senescent TECs

Fourth-generation TECs were collected, the cell concentration was adjusted to 5 × 105 L-1, and 100 μL of cell suspension was inoculated in a 6-well culture plate. When the cell fusion rate was approximately 80%, 200 μmol/L H₂O₂ was added and treated for 72 h, and the supernatant was discarded. Fourth-generation UCSSCs were collected, the cell concentration was adjusted to 5 × 105 L-1, a 100μL cell suspension was inoculated into the Transwell chamber, the Transwell chamber was embedded on the upper layer of the 6-well culture plate, and DMEM/F12 containing 10% foetal bovine serum was used for coculture of cells for 48 hours.

Analysis of ageing-related indexes of TECs after coculture: After coculture, the senescence marker molecules of TECs were analysed, and the cell substructure was observed by fluoroscopic electron microscopy.

Continuous observation of the morphology of cocultured TECs by a Nanolive 4D microscope

P4 generation TECs at a cell concentration of 5000/ml were added to a Nanolive 4D microscope petri dish, and then 1.5 ml DMEM/F12 (containing 10% foetal bovine serum) medium was added and mixed slowly; the cells were then cultured for 24 h. The culture medium was discarded, and the culture medium was discarded after the TECs were treated with 200 μmol/L H2O2 for 72 h. Next, the cell concentration was adjusted to 5000 cells/ml of P4 generation UCSSCs, the Nanolive 4D microscope was started and connected to the computer, the focal length was adjusted to take pictures under the appropriate field of vision for 48 hours, and pictures were taken every 4 min.

DNA methylation and transcriptome sequencing

The TECs of UCSSCs were 1.3x107 in coculture and 1.2x107 in the model group. The cell precipitates were frozen in liquid nitrogen and stored at -80 °C for 24 hours. The whole-genome methylation and transcriptome data were obtained by whole-genome methylation and transcriptome sequencing at Guangzhou Jidiao Biotechnology Co., Ltd.

Analysis of DNA methylation sequencing data

The obtained clean data were mapped to the species reference genome using BSMAP software (version 2.90). Methylated cytosine level was then detected using a custom Perl script and assessed using the correction algorithm described by Lister R, et al. The level of methylation was calculated based on the percentage of methylation of each sequence (CG, CHG, and CHH) in the entire genome, each chromosome, and different regions of the genome. To explain the changes in DNA methylation in TECs, methylation maps of the upstream 1 kb region and gene body were drawn according to the average methylation level of each window. Changes in GC content were screened according to the number of differentially methylated regions (DMRs) (≥ 5) and the absolute value of methylation ratio difference (≥ 0.1). The senescence genes were counted according to GeneOntologyConsortium.

RNA-seq analysis

The transcriptome database was analysed by DESeq2 software, and the mRNAs satisfying the conditions of log2FC > 1.2 and P value < 0.05 were regarded as differentially expressed genes. The differentially expressed genes in ageing TECs and ageing TECs after coculture were counted and analysed by gene function annotation.

Analysis of the relationship between gene methylation and transcriptional level

To calculate the correlation between gene methylation and transcription, the genes were divided into four groups according to the transcription level in RNA-seq data (FPKM ≤ 1 for no transcription, 1 < FPKM ≤ 10 for low transcription, 10 < FPKM ≤ 100 for medium transcription and FPKM > 100 for high transcription). The Pearson correlation coefficients between upstream/gene body methylation levels and corresponding gene expression levels in TECs of the control, model and coculture groups were calculated.

Establishment of the transcription factor-methylation regulatory network

To explore the relationship between specific gene methylation and transcription factor binding, the TFs of ageing-related genes were predicted by CistromeDB, and the TF-DNA regulatory network was constructed by Cytoscape.

Enrichment analysis

Through the Gene Ontology database (GO item in http://www.geneontology.org/)), the number of genes in each item was calculated, and the GO items significantly enriched relative to the genomic background were defined by hypergeometric test. KEGG is the main public pathway-related database (http://www.kegg.jp/kegg/)).

Functional verification

Expression analysis of RAP1 signal pathway molecules RAP1, SRC and ITGB3 For Western blotting analysis, the primary anti-rabbit anti-human RAP1 (1RV 1000, ab14404), SRC (1RV 30000 ab179473 ab109381) and ITGB3 (1RV 1000 ab179473) were all obtained from Abcam, as described above.

Analysis of the relative expression of the ageing regulatory genes NGF, KRT17 and FOXJ1 Total RNA was extracted according to the production manual (Carlsbad Invitrogen Company, California) by RT-QPCR using TRIzol reagent. cDNA was amplified by reverse transcriptase (GoldenstarTMRT6, China) and random primers. According to the SYBR Green Master Mix specification (TsingKe Biotech Co., China), a CFX96TM Real-Time System (Applied 2720) was used for qPCR. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Each experiment was repeated 3 times, and the gene expression level was normalized by the 2-T square method. All qPCR primers are listed in( Table 1).

Immunofluorescence detection of NGF and KI67 expression TECs were collected and fixed with paraformaldehyde, and the fixation solution was removed. Anti-mouse anti-human antibodies against Ki67 and NGF (1R1000 ~ (th) ServiceBio) were added for staining. The nuclei at 15 min were restrained with CY3 goat anti-rabbit DAPI at room temperature for 50 minutes and then sealed with anti-fluorescence quenching sealant. Microscopic examination: The sections were observed under a fluorescence microscope, and images were collected (Nikon DS-U3, Nikon, Japan; DAPI UV excitation wavelength 380 nm, emission wavelength 420 nm, blue light; CY3 excitation wavelength 510-560 nm, emission wavelength 590 nm, red light). The number of positive cells was analysed by ImageJ software (NIH, Bethesda, MD).

Statistical analysis

SPSS 21.0 statistical software was used for analysis, and the data are expressed as the mean ± standard deviation. A t test was used to compare the two groups, and one-way analysis of variance (one-way ANOVA) was used to analyse the significance of differences among the three groups.

TEC: Thymic epithelial cell

ESC: Embryonic stem cell

TSS : Transcription initiation site

TTS : Transcription termination site

cTEC: cortical thymic epithelial cell

mTEC: medullary thymic epithelial cell

UCSSC: Umbilical cord pluripotent-like stem cell

UCMSC: Umbilical cord mesenchymal stem cell

Ethics approval and consent to participate

All experimental protocols were approved by the relevant committees of the 920th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army and the approval number was Lengshen 2021-017 (Section)-01

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by two major Science and Technology Special Projects of Yunnan Science and Technology Program (2018ZF00) and (202002AA100006).

Acknowledgments

We thank everyone on our team for assisting with the preparation of this manuscript.

Sun S, Li JY, Nim HT, Piers A, Ramialison M, Porrello ER, et al. CD90 Marks a Mesenchymal Program in Human Thymic Epithelial Cells In Vitro and In Vivo. Frontiers in Immunology. 2022;13:846281. doi: 10.3389/fimmu.2022.846281.
Gao H, Cao M, Deng K, Yang Y, Song J, Ni M, et al. The Lineage Differentiation and Dynamic Heterogeneity of Thymic Epithelial Cells During Thymus Organogenesis. Frontiers in Immunology. 2022;13:805451. doi: 10.3389/fimmu.2022.805451.
Garcia-Leon MJ, Mosquera M, Cela C, Alcain J, Zuklys S, Hollander G, et al. Abrogation of Notch Signaling in Embryonic TECs Impacts Postnatal mTEC Homeostasis and Thymic Involution. Frontiers in Immunology. 2022;13:867302. doi: 10.3389/fimmu.2022.867302.
Wang HX, Pan W, Zheng L, Zhong XP, Tan L, Liang Z, et al. Corrigendum: Thymic Epithelial Cells Contribute to Thymopoiesis and T Cell Development. Frontiers in Immunology. 2020;11:628464. doi: 10.3389/fimmu.2020.628464.
Shichkin VP, Felli MP, Screpanti I, Antica M. Editorial: Thymus function and aging: A focus on thymic epithelial cells. Frontiers in Immunology. 2022;13:1003490. doi: 10.3389/fimmu.2022.1003490.
Lancaster JN, Keatinge-Clay DE, Srinivasan J, Li Y, Selden HJ, Nam S, et al. Central tolerance is impaired in the middle-aged thymic environment. AGING CELL. 2022;21:e13624. doi: 10.1111/acel.13624.
Tong QY, Zhang JC, Guo JL, Li Y, Yao LY, Wang X, et al. Human Thymic Involution and Aging in Humanized Mice. Frontiers in Immunology. 2020;11:1399. doi: 10.3389/fimmu.2020.01399.
Lepletier A, Chidgey AP, Savino W. Perspectives for Improvement of the Thymic Microenvironment through Manipulation of Thymic Epithelial Cells: A Mini-Review. GERONTOLOGY. 2015;61:504-14. doi: 10.1159/000375160.
Baran-Gale J, Morgan MD, Maio S, Dhalla F, Calvo-Asensio I, Deadman ME, et al. Ageing compromises mouse thymus function and remodels epithelial cell differentiation. eLife. 2020;9. doi: 10.7554/eLife.56221.
Kotasova H, Capandova M, Pelkova V, Dumkova J, Koledova Z, Remsik J, et al. Expandable Lung Epithelium Differentiated from Human Embryonic Stem Cells. Tissue Engineering and Regenerative Medicine. 2022;19:1033-50. doi: 10.1007/s13770-022-00458-0.
Shekari F, Han CL, Lee J, Mirzaei M, Gupta V, Haynes PA, et al. Surface markers of human embryonic stem cells: a meta analysis of membrane proteomics reports. Expert Review of Proteomics. 2018;15:911-22. doi: 10.1080/14789450.2018.1539669.
Chen F, Zhao C, Zhao Y, Li L, Liu S, Zhu Z, et al. The biological characteristics of sheep umbilical cord mesenchymal stem cells. CANADIAN JOURNAL OF VETERINARY RESEARCH-REVUE CANADIENNE DE RECHERCHE VETERINAIRE. 2018;82:216-24.
Garzon I, Alfonso-Rodriguez CA, Martinez-Gomez C, Carriel V, Martin-Piedra MA, Fernandez-Valades R, et al. Expression of epithelial markers by human umbilical cord stem cells. A topographical analysis. PLACENTA. 2014;35:994-1000. doi: 10.1016/j.placenta.2014.09.007.
Naji A, Eitoku M, Favier B, Deschaseaux F, Rouas-Freiss N, Suganuma N. Biological functions of mesenchymal stem cells and clinical implications. CELLULAR AND MOLECULAR LIFE SCIENCES. 2019;76:3323-48. doi: 10.1007/s00018-019-03125-1.
Hoang DM, Pham PT, Bach TQ, Ngo A, Nguyen QT, Phan T, et al. Stem cell-based therapy for human diseases. Signal Transduction and Targeted Therapy. 2022;7:272. doi: 10.1038/s41392-022-01134-4.
Cheng Y, Cao X, Qin L. Mesenchymal Stem Cell-Derived Extracellular Vesicles: A Novel Cell-Free Therapy for Sepsis. Frontiers in Immunology. 2020;11:647. doi: 10.3389/fimmu.2020.00647.
Zhan Y, Wang L, Liu G, Zhang X, Yang J, Pan Y, et al. The Reparative Effects of Human Adipose-Derived Mesenchymal Stem Cells in the Chemotherapy-Damaged Thymus. STEM CELLS AND DEVELOPMENT. 2019;28:186-95. doi: 10.1089/scd.2018.0142.
Kim KH, Lee KA. Metabolic Rewiring by Human Placenta-Derived Mesenchymal Stem Cell Therapy Promotes Rejuvenation in Aged Female Rats. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. 2022;23. doi: 10.3390/ijms23010566.
Tian C, He J, An Y, Yang Z, Yan D, Pan H, et al. Bone marrow mesenchymal stem cells derived from juvenile macaques reversed ovarian ageing in elderly macaques. Stem Cell Research & Therapy. 2021;12:460. doi: 10.1186/s13287-021-02486-4.
Kan RL, Chen J, Sallam T. Crosstalk between epitranscriptomic and epigenetic mechanisms in gene regulation. TRENDS IN GENETICS. 2022;38:182-93. doi: 10.1016/j.tig.2021.06.014.
Song J, VanBuskirk JA, Merbs SL. Regulation of Opsin Gene Expression by DNA Methylation and Histone Acetylation. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. 2022;23. doi: 10.3390/ijms23031408.
Schubeler D. Function and information content of DNA methylation. NATURE. 2015;517:321-6. doi: 10.1038/nature14192.
Li Y, Lei X, Lu H, Guo W, Wu S, Yin Z, et al. Age-Related Changes on CD40 Promotor Methylation and Immune Gene Expressions in Thymus of Chicken. Frontiers in Immunology. 2018;9:2731. doi: 10.3389/fimmu.2018.02731.
Hale LP, Neff J, Cheatham L, Cardona D, Markert ML, Kurtzberg J. Histopathologic assessment of cultured human thymus. PLoS One. 2020;15:e230668. doi: 10.1371/journal.pone.0230668.
Lee EN, Park JK, Lee JR, Oh SO, Baek SY, Kim BS, et al. Characterization of the expression of cytokeratins 5, 8, and 14 in mouse thymic epithelial cells during thymus regeneration following acute thymic involution. Anatomy & Cell Biology. 2011;44:14-24. doi: 10.5115/acb.2011.44.1.14.
Watzek J, Pope SM, Brosnan SF. Capuchin and rhesus monkeys but not humans show cognitive flexibility in an optional-switch task. Scientific Reports. 2019;9:13195. doi: 10.1038/s41598-019-49658-0.
Gupta R, Gupta T, Kaur H, Sehgal S, Aggarwal A, Kapoor K, et al. Cytokeratin (CK5, CK8, CK14) expression and presence of progenitor stem cells in human fetal thymuses. CLINICAL ANATOMY. 2016;29:711-7. doi: 10.1002/ca.22736.
Jung WS, Han SM, Kim SM, Kim ME, Lee JS, Seo KW, et al. Stimulatory effect of HGF-overexpressing adipose tissue-derived mesenchymal stem cells on thymus regeneration in a rat thymus involution model. CELL BIOLOGY INTERNATIONAL. 2014;38:1106-17. doi: 10.1002/cbin.10306.
Liu G, Wang L, Pang T, Zhu D, Xu Y, Wang H, et al. Umbilical cord-derived mesenchymal stem cells regulate thymic epithelial cell development and function in Foxn1(-/-) mice. Cellular & Molecular Immunology. 2014;11:275-84. doi: 10.1038/cmi.2013.69.
Zhu RZ, Li BS, Gao SS, Seo JH, Choi BM. Luteolin inhibits H(2)O(2)-induced cellular senescence via modulation of SIRT1 and p53. KOREAN JOURNAL OF PHYSIOLOGY & PHARMACOLOGY. 2021;25:297-305. doi: 10.4196/kjpp.2021.25.4.297.
Giri S, Takada A, Paudel D, Yoshida K, Furukawa M, Kuramitsu Y, et al. An in vitro senescence model of gingival epithelial cell induced by hydrogen peroxide treatment. Odontology. 2022;110:44-53. doi: 10.1007/s10266-021-00630-3.
Giri S, Takada A, Paudel D, Yoshida K, Furukawa M, Kuramitsu Y, et al. An in vitro senescence model of gingival epithelial cell induced by hydrogen peroxide treatment. Odontology. 2022;110:44-53. doi: 10.1007/s10266-021-00630-3.
Lysek-Gladysinska M, Wieczorek A, Jozwik A, Walaszczyk A, Jelonek K, Szczukiewicz-Markowska G, et al. Aging-Related Changes in the Ultrastructure of Hepatocytes and Cardiomyocytes of Elderly Mice Are Enhanced in ApoE-Deficient Animals. Cells. 2021;10. doi: 10.3390/cells10030502.
Aman Y, Schmauck-Medina T, Hansen M, Morimoto RI, Simon AK, Bjedov I, et al. Autophagy in healthy aging and disease. Nat Aging. 2021;1:634-50. doi: 10.1038/s43587-021-00098-4.
Mangelinck A, Mann C. DNA methylation and histone variants in aging and cancer. International Review of Cell and Molecular Biology. 2021;364:1-110. doi: 10.1016/bs.ircmb.2021.06.002.
Soto-Palma C, Niedernhofer LJ, Faulk CD, Dong X. Epigenetics, DNA damage, and aging. JOURNAL OF CLINICAL INVESTIGATION. 2022;132. doi: 10.1172/JCI158446.
Mohd MN, Aminullah LF, Makpol S. Epigenetic Changes and Its Intervention in Age-Related Neurodegenerative Diseases. CELLULAR AND MOLECULAR NEUROBIOLOGY. 2022;42:577-95. doi: 10.1007/s10571-020-00979-z.
Shah S, Brock EJ, Ji K, Mattingly RR. Ras and Rap1: A tale of two GTPases. SEMINARS IN CANCER BIOLOGY. 2019;54:29-39. doi: 10.1016/j.semcancer.2018.03.005.
Li Y, Song Y, Li P, Li M, Wang H, Xu T, et al. Downregulation of RIG-I mediated by ITGB3/c-SRC/STAT3 signaling confers resistance to interferon-alpha-induced apoptosis in tumor-repopulating cells of melanoma. Journal for ImmunoTherapy of Cancer. 2020;8. doi: 10.1136/jitc-2019-000111.
Salama Y, Heida AH, Yokoyama K, Takahashi S, Hattori K, Heissig B. The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models. Blood Advances. 2020;4:1021-37. doi: 10.1182/bloodadvances.2019001002.
Li H, Yu T, Ma Y, Wang H. [Expression of Laminin gene family in porcine pluripotent stem cells]. 生物工程学报. 2017;33:1304-14. doi: 10.13345/j.cjb.170101.
Sendzikaite G, Kelsey G. The role and mechanisms of DNA methylation in the oocyte. Essays in Biochemistry. 2019;63:691-705. doi: 10.1042/EBC20190043.
Varley KE, Gertz J, Bowling KM, Parker SL, Reddy TE, Pauli-Behn F, et al. Dynamic DNA methylation across diverse human cell lines and tissues. GENOME RESEARCH. 2013;23:555-67. doi: 10.1101/gr.147942.112.
Heberle E, Bardet AF. Sensitivity of transcription factors to DNA methylation. Essays in Biochemistry. 2019;63:727-41. doi: 10.1042/EBC20190033.
Lee HW, Kim SM, Shim NR, Bae SK, Jung IG, Kwak JY, et al. Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution. Regul Pept. 2007;141:86-95. doi: 10.1016/j.regpep.2006.12.015.
Lee HW, Na YJ, Jung PK, Kim MN, Kim SM, Chung JS, et al. Nerve growth factor stimulates proliferation, adhesion and thymopoietic cytokine expression in mouse thymic epithelial cells in vitro. Regul Pept. 2008;147:72-81. doi: 10.1016/j.regpep.2008.01.004.

Tables 1 to 7 are available in the Supplementary Files section.

No competing interests reported.

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Umbilical cord subtotipotent-like stem cells reverse the ageing of thymic epithelial cells by inducing reprogramming of the whole-genome methylation and transcription profile

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