Induction of Salivary Gland-Like Tissue by Induced Pluripotent Stem Cells In Vitro

Abstract

Background:

To investigate the in vitro induction of salivary gland-like tissue by ips cells in an interferon regulatory factor 6 (IRF6) overexpression and parotid conditioned medium environment.

Methods:

Urine-derived ips cells were isolated, identified, transfected with IRF6 and cultured in parotid conditioned medium to induce ips cells into salivary gland differentiation, morphological changes of ips cells were observed, CCK-8 was used to determine the cell proliferation efficiency and transcriptome sequencing was used to detect the expression of genes related to parotid gland formation.

Results:

Immunofluorescence staining showed that the isolated ips cells were positive for NANOG, SSEA4 and OCT4 and had embryonic-like stem cell characteristics; CCK-8 showed that there was no statistical difference in the proliferation efficiency between the IRF6⁺ induced group and the simple induced group after induction of ips cells into salivary glands. The results of transcriptome sequencing showed that there were a total of 643 differentially expressed genes, including 365 up-regulated genes and 278 down-regulated genes in the IRF6⁺ induced group compared to the blank control group, and the salivary gland related genes HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM were all highly expressed in the IRF6⁺ induced group.

Conclusion:

IRF6 promotes salivary gland differentiation in urine-derived iPSCs, and its mechanism of promoting differentiation may be that IRF6 upregulates the expression of HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM to promote epithelial differentiation.

Introduction

Oral health is closely related to many important organ diseases of the body and to the quality of human existence. Among the various factors that affect oral health, saliva is one of the most important. Irreversible destruction of salivary gland tissue, often caused by radiotherapy and surgery for head and neck tumours, as well as by Schegren's syndrome, and low salivary secretion can lead to severe xerostomia, which in turn can lead to a variety of oral diseases and even affect general health [1]. Regeneration of salivary gland tissue may be the only way to treat the loss of salivary gland tissue function. There are currently no salivary gland stem cells with both secretory and tissue regeneration capabilities that have been accepted by the academic community, and the challenge of obtaining sufficient salivary gland cells to achieve therapeutic quantities in tissue regeneration studies remains [2, 3].

Induced pluripotent stem cells (IPSCs) are clones of cells that have been reprogrammed from somatic cells and induced to resemble embryonic stem cells (ESCs). iPSCs are totipotent stem cells similar to ESCs in gene expression, pluripotent differentiation and epigenetics. They are a valuable stem cell resource for clinical tissue repair and regeneration research, and are widely used for drug screening, drug development and mechanistic studies of specific diseases [4].

The use of totipotent or pluripotent stem cells to regenerate salivary organs with intact structure and function requires an in-depth study of the developmental mechanisms of salivary gland tissue. Interferon regulatory factor 6 (IRF6) is a transcription factor that regulates the exit of cell cycle and acts on ectodermal tissue differentiation and stimulation of interferon [5]^. Mutations in IRF6 increase the risk of type II cleft lip and palate and Van der Wound syndrome [6]. Recent studies have found that tissue-specific IRF6 deficiency leads to salivary gland dysfunction [7]. Several investigators have studied the pathology of patients with Van der Wound syndrome and found ectopic hyperplasia of some of the minor salivary glands [8, 9]. It has also been found that IRF6 knockout transgenic mice exhibit reduced salivary secretion, increased mucus acidity and hypoplastic salivary glands, and these mice develop severe dental caries on a high sugar diet [10], which provide a reasonable explanation for the suspicion that cleft lip and palate patients often have very severe caries. Saliva is derived from the ectoderm and the p53 gene is essential for the development of ectodermal structures. There is now ample evidence that IRF6 is closely related to the p53 gene and that p53 is a positive regulator of IRF6 and contributes to its expression [11]. We therefore have reason to believe that IRF6 is closely associated with salivary gland development. Intervention of IRF6 gene expression, combined with the pluripotent regenerative capacity of iPSCs, could potentially be one of the viable options for salivary gland tissue regeneration.

Materials and methods

This study was conducted in accordance with the Declaration of Helsinki and the required ethical approval was granted by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University. The patients were informed of the process and risks involved in the collection and use of the specimens in this study, and they or their immediate family members signed an informed consent form.

Isolation and identification of urine-derived iPSCs

Urine cell reprogramming to obtain iPSCs

Enough urine sample was taken and urine cells were collected and amplified to obtain a sufficient amount of urine cells with a cell volume of approximately 5.0 × 10⁶ for the induction of iPSCs. Cells were electrotransfected with 10 μg of free plasmid mixture OCT3/4, SOX2, KLF4, c-MYC, LIN28, TP53 shRNA (Addgene, Cambridge, MA, USA) using a cell transfection kit (Amaxa Nucleofector Kit, Lonza, Basel, Switzerland) via a single-well cell nuclear transfection system (Nucleofector 2b Device, Lonza, Basel, Switzerland) [12]. The transfected cells were placed on 12-well plates coated with Matrigel (BD Biosciences, Bedford, MA, USA) and proliferated in reprogramming medium (Reproeasy hiPSC Reprogramming Kit, Sabe Biologics, China). When ESCs-like (iPSCs) clones were found to form, the clones were transferred to Matrigel-coated 6-well plates and proliferated using E8 medium (TeSR-E8 Complete Kit, STEMCELL Technologies, Vancouver, Canada) to form stable clones of iPSCs.

Cellular immunofluorescence assay for pluripotent markers of urine cell-derived iPSCs

The slides with cells were washed in PBS in culture plates, fixed with 4% paraformaldehyde for 15 min, permeabilised with 0.5% Triton X-100 for 20 min, washed in PBS, closed with goat serum for 30 min at room temperature, added primary antibody to incubate overnight at 4 °C, washed in PBS, incubated with fluorescent secondary antibody for 1 h at 37 °C, washed in PBS and observed under a fluorescence microscope. Immunofluorescence staining for embryonic stem cell specific antigens NANOG, SSEA4, OCT4 (Abcam, Cambridge, UK) was performed on the isolated iPSCs.

Isolation of primary parotid cells and preparation of conditioned media

Parotid tissue sampling

Parotid tissue samples were obtained from parotidectomy specimens in the Department of Stomatology, Beijing Friendship Hospital, Capital Medical University (IRB number: 2020-P2-209-02). Both patients had preoperative pathology reports and were diagnosed with pleomorphic adenoma of the parotid gland. The sampling required intraoperative excision of the tumour from the parotid area in a standardised procedure that should include at least 0.5 cm of normal tissue lateral to the tumour margin. The normal parotid tissue surrounding the surgical specimen should be excised under aseptic conditions.

Primary culture of parotid cells

The following steps needed to be performed under aseptic conditions. The specimens were isolated and washed 2–3 times in PBS under aseptic conditions, and the parotid tissue was cut into as small pieces as possible, approximately 1 mm in diameter, using ophthalmic scissors. The tissue pieces were placed in a culture dish with sterile forceps, spaced approximately 0.5–1.0 cm apart, and left to stand for 10 min. DMEM complete medium containing 10% FBS and 1% double antibiotics was gently and slowly added along the side walls of the dish and placed in a cell culture incubator. Passage was possible when cell creep was observed around the tissue and local fusion was greater than about 30%.

Collection of conditioned media

From the second day of primary culture of parotid cells, the culture was collected on alternate days in a 15 ml centrifuge tube and DMEM complete medium containing 10% FBS and 1% double antibiotics was added to the culture dish. The collected supernatant was centrifuged at 800 rpm for 4 min and the supernatant was collected and stored at 4 °C and used as conditioned medium for subsequent experiments.

Transfection of iPSCs with IRF6-overexpressing lentiviral vector

The lentiviral vector GV416 (GKN, Shanghai, China) with IRF6 was infected with iPSCs for 47–72 h. The infection efficiency of the cells was observed under a fluorescence microscope (DM5000B, Leica, Germany) and when > 80%, the E8 medium containing 1 μg/ml puromycin was replaced for drug screening until all cells in the control wells died and IRF6⁺ -iPSCs were obtained. The cells were cultured in E8 medium and subcultured in preparation for subsequent experiments.

Salivary gland differentiation of IRF6⁺-iPSCs

IRF6⁺-iPSCs (IRF6⁺ induced group) and iPSCs (simple induced group) were cultured in Matrigel-coated 24-well plates with 1 ml of conditioned medium and changed every other day. The iPSCs were routinely cultured in 24-well plates using E8 medium (blank control group).

Cell growth curve by CCK-8 assay

The plates were incubated for 4 h at 37 °C in a constant temperature incubator with 10 μl of CCK-8 solution (CCK-8 Cell Proliferation and Cytotoxicity Assay Kit, Solarbio, Beijing, China) added to the wells on days 1, 2, 3, 4 and 5 respectively. The absorbance values of each group at 450 nm were measured by ELISA and the corresponding growth curves were plotted.

Transcriptome sequencing

Extraction of RNA

1 ml of Trizol was added to the Petri dish, mixed well by blowing, 0.2 ml of chloroform was added, shaken well, the supernatant was taken by centrifugation at 12000 g 4 °C for 15 min, 0.5 ml of isopropanol was added, the supernatant was discarded by centrifugation at 12000 g 4 °C for 10 min, 1 ml of 75% ethanol was added to wash the precipitate, the supernatant was discarded by centrifugation at 7500 g 4 °C for 5 min, the precipitate was dried and dissolved with DEPC H₂O. RNase-Free treated consumables were used throughout to prevent contamination by exogenous enzymes.

Library construction

Steps included primer annealing, reverse transcription into cDNA plus switch oligo; synthesis of complementary strands; DNA damage repair and end repair, magnetic bead purification.

Sequencing, followed by bioinformatics analysis

The results of the sequencing were obtained through analysis on the BMKCloud cloud platform (www.biocloud.net).

Statistical methods

This study used SPSS 20.0 statistical software for data processing, and the measurement data were expressed as mean ± standard deviation (x ± s). Count data were expressed as percentages (%). One-way analysis of variance (ANOVA) was used for comparisons between multiple groups that obeyed a normal distribution, with a post hoc test as LSD; non-parametric tests were used for comparisons between groups that did not obey a normal distribution. Count data were analyzed by chi-square test. p < 0.05 was considered a statistically significant difference. Statistical analysis was performed and graphs were drawn using Graphpad Prism software.

Results

Parotid cell primary culture and collection of conditioned medium

Parotid primary cells were successfully obtained using the explant tissue culture method. On day 3 of primary culture, a few cells were seen crawling out from the edge of the tissue block. The cells were mainly polygonal, pavement-like, long spindle-shaped or spindle-shaped, with a small number of irregularly shaped vacuolated mast cells; on day 7, the cell consistency increased and the proportion of long spindle-shaped cells increased, radiating along the edge of the tissue block; on day 10, the number of cells at the edge of the tissue block increased further, with a large number of long spindle-shaped mesenchymal stem cells and a small number of polygonal epithelial-like cells. The fusion of cells increased, the gap became smaller, and some irregular cells showed apoptosis. The consistency of the cells increased after passaging, and after P4 they were mainly mesenchymal stem cells under the microscope (Fig. 1). A sufficient amount of conditioned medium was purified by collecting the supernatant of parotid primary cells.

Fig. 1.

Primary culture of parotid cells (100×). A cell morphology was mainly spindle and polygonal; B increased proportion of spindle-shaped mesenchymal stem cells, with a few vacuolated cells visible

Cellular immunofluorescence assay for pluripotent markers of urine-derived iPSCs

In this study, urine cells were isolated and cultured and then reprogrammed for induction, and all were successfully transformed into iPS clones. Immunofluorescence staining results indicated positive staining for the embryonic stem cell specific antigens NANOG, SSEA4 and OCT4 (Fig. 2).

Fig. 2.

A–C Cellular immunofluorescence detection of stem cell markers for urine cell-derived iPSCs (100×). Positive staining results for embryonic stem cell specific antigens NANOG (A), SSEA4 (B) and OCT4 (C)

Cell fluorescence observation after IRF6 lentiviral vector infection of iPSCs

A more obvious green fluorescence could be observed under fluorescence microscope after 72 h of IRF6 lentivirus infection of iPSCs (Fig. 3), with high infection efficiency and good cell status. It indicated that the GFP fluorescence gene expression was normal, the target gene transfection was normal, and the lentiviral vector was more efficient in infecting iPSCs with good infection efficiency.

Fig. 3.

iPSCs (50×) were infected with lentiviral vector-mediated overexpression of IRF6. 72 h later a clear green fluorescence was visible, GFP expression was normal and the infection efficiency was > 80%

Cell growth curve by CCK-8 assay

Comparing the changes in cell proliferation activity in the three groups of culture conditions, according to the following figure (Fig. 4), no significant changes were seen in the OD values of the cells in each group on day 1, and from the second day onwards the cell proliferation activity of the IRF6⁺ induced group and the simple induced group was significantly lower (p < 0.05) than that of the blank control group.

Fig. 4.

Growth curves for different treatments (CCK8 assay)

Microscopic observation of salivary gland differentiation morphology of IRF6⁺ -iPSCs

To study the differentiation of iPSCs into salivary glands and the effect of IRF6 gene on them, we used conditioned medium as the induction medium for IRF6⁺-iPSCs and iPSCs, and iPSCs⁺ E8 medium as a blank control. The two groups of cells in the conditioned medium produced more obvious changes in morphology, showing epithelial differentiation, as evidenced by a widening of the cell gap, a change from round to polygonal or shuttle-shaped, and a certain consistency in morphology, an increase in cell volume, and a decrease in the proportion of nucleoplasm. Differentiation of some cells was observed on day 2 of the conditioned medium culture, and by day 4 differentiation could be generally observed under the microscope (Figs. 5 and 6). The iPS cells showed directional differentiation towards salivary gland tissue, and after 3 days a salivary gland alveolar cell-like morphology (Fig. 7), salivary gland alveoli and duct-like structures (Fig. 7) were observed, and the cell supernatant was positive for amylase activity assay (Table 1).

Fig. 5.

A–I Micrographs of iPSCs differentiation into salivary glands at different times (100×). In A/D/G: IRF6⁺ induced group (day0/day2/day4) and B/E/H: iPSCs simple induced group (day0/day2/day4), differentiation was observed on the second day of induction, showing a widening of the cell gap, from round to polygonal or spindle-shaped with some consistency in morphology. Differentiation was generally observed microscopically by day 4. C/F/I: no differentiation was observed in the iPSCs blank control group (day0/day2/day4)

Fig. 6.

A–I Micrographs of iPSCs differentiation into salivary gland at different magnifications (day4). In A/B/C: IRF6⁺ induced group (50×/100×/400×) and D/E/F: iPSCs simple induced group (50×/100×/400×), epithelial differentiation was commonly observed microscopically on day 4, as evidenced by a widening of the cell gap, from round to polygonal or spindle-shaped, and an increase in cell volume with some consistency in morphology. The cells in the G/H/I: iPSCs blank control group (50×/100×/400×) did not show any differentiation

Fig. 7.

iPSCs differentiated into alveolar-like structures. Salivary gland alveolar-like morphology could be observed after 3 days. A: IRF6⁺ induced group; B: iPSCs simple induced group. iPSCs differentiated into duct-like structures. Salivary gland duct-like morphology was observed after 3 days. C: IRF6⁺ induced group; D: iPSCs simple induced group

Table 1.

Comparison of amylase assays in the supernatant of the three groups of cells

	IRF6+ induced group	iPSCs simple induced group	Blank control group
Amylase U/L	1552	1320	0

Analysis of differentially expressed genes

Differential analysis was performed using edgeR based on the relative high and low gene expression levels between the IRF6⁺ induced group and the blank control group of iPSCs. As shown in Table 2, there were 643 differentially expressed genes, including 365 up-regulated genes and 278 down-regulated genes, and Fig. 8 showed the scatter plot of differentially expressed genes. Fold Change ≥ 2 and P-value < 0.05 were used as screening criteria in the differentially expressed gene detection process. The Fold Change was the ratio of expression between the two groups and p-value was the screening index for the significance of differentially expressed genes.

Table 2.

Statistics on the number of differentially expressed genes between the IRF6⁺ induced group and the iPSCs blank control group

DEG Number	Up-regulated	Down-regulated
643	365	278

DEG Number: number of differentially expressed genes; up-regulated: number of up-regulated genes

Fig. 8.

Differentially expressed genes in the IRF6⁺ induced group and the iPSCs blank control group. Each point in the graph represented a gene. The horizontal coordinate was the A value: log2(CPM), the logarithm of the mean expression in the two samples; the vertical coordinate was the M value: log2(FC), the logarithm of the fold difference in gene expression between the two samples, used to measure the magnitude of the expression difference. The green dots in the graph represented down-regulated differentially expressed genes (p < 0.05), the red dots represented up-regulated differentially expressed genes (p < 0.05) and the black dots represented non-differentially expressed genes (p > 0.05). down-regulated: number of down-regulated genes

We clustered the 40 genes with the greatest differential expression among these up- and down-regulated genes and plotted the gene heatmap (Fig. 9), and found that seven marker genes of the salivary gland were all highly expressed, namely HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM, respectively.

Fig. 9.

Clustering analysis of some of the differentially expressed genes in the IRF6⁺ induced group and the iPSCs blank control group. The horizontal coordinates represented the IRF6⁺ induced group and the iPSCs blank control group respectively, and the vertical coordinates represented the differentially expressed genes and the clustering results of these genes. Different rows in the graph represented different genes. The colours represented the expression levels of the genes in the samples, log2 (CPM + 1e-6)

KEGG (Kyoto Encyclopedia of Genes and Genomes) is a systematic analysis of gene function and genome information database. It helps researchers to study genes and expression information as a whole network. We further carried out KEGG annotation analysis on differentially expressed genes (shown as in Fig. 10), which helped to further interpret the function of genes. We took 'Pathway' in the KEGG database as a unit, applied hypergeometric test to carry out Pathway significance enrichment analysis, and fiound the Pathway that is significantly enriched in differentially expressed genes compared with the entire gene background. The results of enrichment analysis of the differentially expressed genes KEGG pathway were shown in Fig. 11. The figure showed the top 20 pathways with the least significant Q value.

Fig. 10.

KEGG classification diagram of differentially expressed genes between IRF6⁺ induced group and the blank control group of iPSCs. The y-axis was the name of the KEGG metabolic pathway, and the x-axis was the number of genes annotated to the pathway and their proportion to the total number of genes annotated

Fig. 11.

KEGG pathway enrichment analysis of DEGs between IRF6⁺ induced group and the blank control group of iPSCs. Each circle in the figure represents a KEGG pathway, the ordinate indicates the name of the pathway, and the abscissa is the Enrichment Factor, which indicates the proportion of genes annotated to a pathway among the differential genes and the genes annotated to the pathway among all genes The ratio of the ratio. The larger the enrichment factor, the more significant the enrichment level of differentially expressed genes in this pathway. The color of the circle represents the q value, which is the P value after multiple hypothesis test correction. The smaller the qvalue, the more reliable the significance of the enrichment of differentially expressed genes in the pathway; the size of the circle indicates the number of genes enriched in the pathway, the circle The larger the gene, the more genes

Analysis of differentially expressed transcripts

In order to determine the similarity between the differentiated salivary gland cells induced by IRF6 + group and Salivary Gland- like Cells, by comparing different databases, the differentially expressed transcripts between the IRF6 + induction group and iPSCs blank control group and parotid primary cells were analyzed (Table 3). In addition, the annotation results of differentially expressed transcripts KEGG were classified according to the types of pathways in KEGG. The classification diagram was shown in Fig. 12, and the pathway enrichment analysis of differentially expressed transcripts was shown in Fig. 13.

Table 3.

KEGG pathway enrichment analysis of differential expression transcript among IRF6⁺ induced group, iPSCs blank control group and Salivary Gland like-Cells

	Total	COG	GO	KEGG	KOG	NR	Pfam	Swiss-Prot	Eggnog
IRF6+ induced group vs. iPSCs blank control group	303	101	231	203	196	301	243	294	287
IRF6+ induced group vs. Salivary Gland-like Cells	1796	533	1161	1323	1103	1791	1569	1755	1736

Total: the number of differentially expressed transcripts annotated; the third column to the last column indicated the number of differentially expressed transcripts annotated by each functional database

Fig. 12.

KEGG classification map of DEGs among IRF6⁺ induced group, iPSCs blank control group, and Salivary Gland like-Cells. A IRF6⁺ induced group vs iPSCs blank control group; B IRF6⁺ induced group vs Salivary Gland like-Cells; the ordinate is the name of the KEGG metabolic pathway, and the abscissa is the number of transcripts annotated to the pathway and its percentage The proportion of the total number of annotated transcripts

Fig. 13.

KEGG pathway enrichment analysis of differential expression transcript among IRF6⁺induced group, iPSCs blank control group and Salivary Gland like-Cells. A IRF6⁺ induced group vs iPSCs blank control group; B IRF6⁺ induced group versus Salivary Gland like-Cells; each circle in the figure represents a KEGG pathway, the ordinate indicates the name of the pathway, and the abscissa is the Enrichment Factor. Indicates the ratio of the proportion of transcripts annotated to a pathway in the differential transcripts to the proportion of transcripts annotated to the pathway in all transcripts. The larger the enrichment factor, the more significant the enrichment level of differentially expressed transcripts in this pathway. The color of the circle represents the qvalue, which is the P value after multiple hypothesis testing correction. The smaller the q value, the more reliable the significance of the enrichment of differentially expressed transcripts in the pathway; the size of the circle indicates the number of enriched transcripts in the pathway, The larger the circle, the more transcripts

Discussion

Irreversible damage to the gland is often caused by post-operative salivary gland tumours and radiotherapy to the head and neck, as well as certain autoimmune diseases, often manifesting as dry mouth, painful and easily damaged mucosa, which in turn leads to abnormal taste, halitosis, chewing, swallowing and speech difficulties. In addition, the reduction in total saliva secretion reduces the local antimicrobial capacity, buffering capacity, self-cleaning capacity and dental tissue remineralization ability of the oral cavity, which will lead to rapid progression of caries and loss of hard dental tissue, greatly reducing the patient's quality of life and increasing medical costs. For primary and spontaneous loss of salivary secretion, clinical treatment is currently based on medication to promote salivary secretion and salivary substitutes. However, there is a lack of effective treatment options for patients with complete destruction of the parenchymal cells of the gland. With the development of stem cell technology and tissue engineering, stem cell therapy may be one of the effective means to address salivary gland damage in the future. This project combines stem cell technology and genetic engineering in an attempt to map out the conditions for differentiation of iPSCs into salivary glands and the associated mechanisms.

The discovery of iPSCs was one of the major milestones in stem cell research, for which his discoverer Shinya Yamanaka was awarded the Nobel Prize in Physiology or Medicine in 2012. iPSCs are now considered to be a reliable stem cell technology with promising applications. Compared to other stem cells, iPSCs avoid many of the ethical and legal barriers to the application of ESCs in research. They can be induced from a wide range of somatic cells, are widely available, have the advantage of being easily accessible, and are less ethically controversial than ESCs. They also avoid the problem of immune rejection because they carry the patient's own genes. In addition, iPSCs have a differentiation potential similar to that of ESCs and can theoretically be induced into all types of adult cells, and they have some characteristic genetic memory that facilitates differentiation into the tissue from which they originate [4, 13]. Previously study had identified KIT + epithelial cells as the marker to generate new mouse salivary gland, also, some stuides found that nonepithelial cells (like Bone Marrow derived mesenchymal stem cells or human adipose-derived mesenchymal stem cells) can be used to regenerate mouse salivary gland [14, 15]. Hence the tissue engineering strategies are important potential therapy treatment. The source of the iPSCs used in this study was urine cells, mainly renal epithelial cells which is easy to obtain without pain on patients. And we identified a new target for stimulation the generation of salivary gland. The urine cells were extracted, amplified and reprogrammed to induce stable passages of iPSCs. Cellular immunofluorescence staining experiments showed positive staining for ESCs-specific antigens NANOG, SSEA4 and OCT4, demonstrating that they had ESCs-like characteristics and their differentiation potential was pluripotent. The iPSCs used in this study were easily accessible, taken from the autologous body, could be extracted directly from urine. The extraction process was non-invasive, and there was no immune rejection. Given the advantages described above, urine-derived iPSCs are more suitable as seed cells for adult salivary gland differentiation than ESCs or MSCs.

The salivary gland is an exocrine gland, derived from the ectoderm, whose main function is to produce saliva and digestive enzymes that break down various nutrients [16, 17]. The salivary gland contains glandular epithelium cells as well as vessel element, and its development is the result of a combination of epithelial and mesenchymal action, regulated by a variety of genes, proteins, cytokines and signalling pathways [18]. In this study, differentiation in the direction of salivary glands and formation of alveolar-like and duct-like tissues were observed microscopically in both the IRF6⁺-induced and simple induced groups. Their histological manifestations were significantly different from those of the blank control group, and the cell supernatant was positive for amylase assay, indicating that iPSCs differentiated toward salivary glands. In subsequent experiments, by sequencing the genes of IRF6⁺ induced group and the blank control group, a total of 643 genes were found to be differently expressed, of which 365 were up-regulated and 278 were down-regulated. The 40 genes with the greatest differential expression in these up-regulated and down-regulated genes, respectively, were further analyzed, and seven marker genes of salivary glands were screened, namely HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM. Previous investigators have demonstrated that HAPLN1, MSX2, ANXA1 and CYP11A1 are expressed in the salivary gland [19–22], CCR2 is expressed in the salivary gland epithelium [23] and HES1 and LUM are expressed in the ductal epithelium of the salivary gland [24, 25]. This further supports the previous hypothesis that IRF6 plays a role in the differentiation of iPSCs into salivary glands.

The growth curves of the three groups were evaluated by CCK8 assay, and the results showed that the growth curves of the IRF6⁺ induced group and the simple induced group were both lower than those of the blank control group, and there was no significant difference in the growth curves of the first two groups. Thus, we speculated that the most important factor affecting the cell proliferation rate in this experiment was the difference of medium composition, and IRF6 did not affect the cell proliferation rate during the induction of adult salivary glands.

There is now a wealth of research demonstrating that a variety of genes and pathways are involved in salivary gland secretion, but gene therapy is not just about upregulating or suppressing a particular gene. Salivary gland secretion is the result of the interaction of multiple factors, and most of the studies are currently limited to the effects of individual genes on secretion. As for the mechanism of action of gene–gene, protein-gene and certain small molecule compounds on salivary gland secretion, few studies have been reported, and further research is needed to investigate the principle of salivary gland gene regulation.

In conclusion, IRF6 promoted the differentiation of salivary glands from urine cell-derived iPSCs and had no inhibitory effect on cell proliferation. The mechanism of its promotion of differentiation may be that IRF6 up-regulated the expression of HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM to promote epithelial differentiation.

Declarations

Conflict of interest

All of the authors had no any personal, financial, commercial, or academic conflicts of interest separately.

Ethical statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of our Hospital (IRB no. 2020-P2-209-02).