Human Periodontal Mesenchymal Stem Cells Research Articles

ZEB1 interferes with human periodontal ligament stem cell proliferation and differentiation.

Periodontitis can eventually contribute to tooth loss. Zinc finger E-box binding homeobox 1 (ZEB1) is identified as overexpressed in the gingival tissue of mice with periodontitis. This study is designed to decipher the mechanism of ZEB1's involvement in periodontitis. Human periodontal mesenchymal stem cells (hPDLSCs) were exposed to LPS to mimic the inflammation in periodontitis. Following ZEB1 silencing, FX1 (an inhibitor of Bcl-6) treatment or ROCK1 overexpression, cell viability, and apoptosis were analyzed. Alkaline phosphatase (ALP) staining, Alizarin red staining, RT-qPCR, and western blot were performed to evaluate osteogenic differentiation and mineralization. hPDLSCs were processed for luciferase reporter assay and ChIP-PCR to confirm the association between ZEB1 and ROCK1. The induction of ZEB1 silencing resulted in reduced cell apoptosis, enhanced osteogenic differentiation, and mineralization. Nevertheless, these effects were significantly blunted by FX1. ZEB1 was confirmed to bind to the promoter sites of ROCK1 and regulate the ROCK1/AMPK. Whereas ROCK1 overexpression reversed the effects of ZEB1 silencing on Bcl-6/STAT1, as well as cell proliferation and osteogenesis differentiation. hPDLSCs displayed decreased proliferation and weakened osteogenesis differentiation in response to LPS. These impacts were mediated by ZEB1 regulating Bcl-6/STAT1 via AMPK/ROCK1.

Characterization and Study of Gene Expression Profiles of Human Periodontal Mesenchymal Stem Cells in Spheroid Cultures by Transcriptome Analysis.

A spheroid is known as a three-dimensional culture model, which better simulates the physiological conditions of stem cells. This study is aimed at identifying genes specifically expressed in spheroid-cultured human periodontal ligament mesenchymal stem cells (hPDLMSCs) using RNA-seq analysis to evaluate their functions. Transcriptome analysis was performed using spheroid and monolayer cultures of hPDLMSCs from four patients. Cluster and Gene Ontology analyses revealed that genes involved in cell-cell adhesion as well as the G2/M and G1/S transitions of mitotic cell cycles were strongly expressed in the monolayer culture group. However, genes involved in the negative regulation of cell proliferation, histone deacetylation, and bone morphogenetic protein signaling were strongly expressed in the spheroid culture group. We focused on the transcription factor nuclear receptor subfamily 4 group A member 2 (NR4A2) among the genes that were strongly expressed in the spheroid culture group and analyzed its function. To confirm the results of the transcriptome analysis, we performed real-time polymerase chain reaction and western blotting analyses. Interestingly, we found that the mRNA and protein expressions of NR4A2 were strongly expressed in the spheroid-cultured hPDLMSCs. Under osteogenic differentiation conditions, we used siRNA to knock down NR4A2 in spheroid-cultured hPDLMSCs to verify its role in osteogenesis. We found that NR4A2 knockdown significantly increased the levels of mRNA expression for osteogenesis-related genes alkaline phosphatase (ALP), Osteopontin (OPN), and type 1 collagen (COL1) (Student's paired t-test, p < 0.05). ALP activity was also significantly increased when compared to the negative control group (Student's paired t-test, p < 0.05). Additionally, spheroid-cultured hPDLMSCs transfected with siNR4A2 were cultured for 12 days, resulting in the formation of significantly larger calcified nodules compared to the negative control group (Student's paired t-test, p < 0.05). On the other hand, NR4A2 knockdown in hPDLMSC spheroid did not affect the levels of chondrogenesis and adipogenesis-related genes under chondrogenic and adipogenic conditions. These results suggest that NR4A2 negatively regulates osteogenesis in the spheroid culture of hPDLMSCs.

Effect of TNF-α on the proliferation and osteogenesis of human periodontal mesenchymal stem cells.

The aim of the present study was to investigate the effect of tumor necrosis factor-α (TNF-α) on the proliferation and osteogenesis of human periodontal mesenchymal stem cells (hPDLSCs). Antigen expression in hPDLSCs was detected by flow cytometry. hPDLSCs were divided into four groups: A control group with no TNF-α treatment, and three experimental groups treated with 0.1, 1 and 10 ng/ml TNF-α, respectively. The effect of TNF-α on proliferation of hPDLSCs in vitro was detected using a Cell Counting Kit-8 assay. Differentiation into an osteogenic lineage was detected by alkaline phosphatase sand alizarin red staining, and the mRNA and protein expression levels of runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and type I collagen (Col-I) were detected using reverse transcription-quantitative PCR and western blot respectively. Following treatment with 10 ng/ml TNF-α, proliferation was significantly increased compared with an untreated control group (P<0.01). Additionally, there was a significant inhibition of alkaline phosphatase enzyme activity, alizarin red mineralization node size, and in the gene and protein expression levels of osteogenic differentiation markers, including Runx2, OCN and COL-I (all, P<0.05). Taken together, the results indicated that treatment with 10 ng/ml TNF-α promoted the proliferation of hPDLSCs in vitro and inhibited osteogenic differentiation of hPDLSCs, providing an experimental basis for regulation of hPDLSC-mediated periodontal tissue regeneration.

Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients.

Periodontitis impairs the osteogenic differentiation of human periodontal mesenchymal stem cells (hPDLSCs), but the underlying molecular mechanisms are still poorly understood. Long noncoding RNAs (lncRNAs) have been demonstrated to have significant roles under both physiologic and pathological conditions. In this study, we performed comprehensive lncRNA profiling by lncRNA microarray analysis and identified a novel lncRNA, osteogenesis impairment-related lncRNA of PDLSCs from periodontitis patients (lncRNA-POIR), the expression of which was significantly decreased in PDLSCs from periodontitis patients (pPDLSCs) and was upregulated by osteogenic induction. To study the functions of lncRNA-POIR, we prepared cells with overexpression and knockdown of lncRNA-POIR and found that lncRNA-POIR positively regulated osteogenic differentiation of hPDLSCs and pPDLSCs both in vitro and in vivo. Using quantitative real-time PCRs (qPCRs) and luciferase reporter assays, we demonstrated that lncRNA-POIR may act as a competing endogenous RNA (ceRNA) for miR-182, leading to derepression of its target gene, FoxO1. In this process, lncRNA-POIR and miR-182 suppress each other and form a network to regulate FoxO1. FoxO1 increased bone formation of pPDLSCs by competing with TCF-4 for β-catenin and inhibiting the canonical Wnt pathway. Finally, inflammation increases miR-182 expression through the nuclear factor-κB pathway, and the miR-182 overexpression in the inflammatory microenvironment resulted in an imbalance in the lncRNA-POIR-miR-182 regulatory network. In conclusion, our results provide novel evidence that this lncRNA-miRNA (microRNA) regulatory network has a significant role in osteogenic differentiation of pPDLSCs and that it has potential as a therapeutic target in mesenchymal stem cells during inflammation.