Analysis of the phosphoproteome in human dental follicle cells during osteogenic differentiation.

Dental follicle cells (DFCs) are osteogenic progenitor cells and are well suited for molecular studies of differentiation of alveolar osteoblasts. A recent study examined the metabolism in DFCs during osteogenic differentiation and showed that energy metabolism is increased after 14 days of differentiation (mid phase). However, previous studies have examined proteomes at early (2 h, 24 h) or very late (28 days) stages of differentiation, but not during the phase of increased metabolic activity. In this study, we examined the phosphoproteome at the mid phase (14 days) of osteogenic differentiation. Analysis of DFC phosphoproteomes showed that during this phase of osteogenic differentiation, proteins that are part of signal transduction are significantly regulated. Proteins involved in the regulation of the cytoskeleton and apoptosis were also increased in expression. As osteogenic differentiation induced oxidative stress and apoptosis in DFCs, the oxidative stress defense protein, catalase, was also upregulated during osteogenic differentiation, which supports the biomineralization of DFCs. In summary, this study revealed that during the middle phase (14 days) of osteogenic differentiation, processes in DFCs related to the control of cell organization, apoptosis, and oxidative stress are regulated.

The SUMO-specific isopeptidase SENP3 regulates MLL1/MLL2 methyltransferase complexes and controls osteogenic differentiation.

The ubiquitin-like SUMO system regulates gene expression, but the molecular insights into this process are incomplete. We show that the SUMO-specific isopeptidase SENP3 controls H3K4 methylation by regulating histone-modifying SET1/MLL complexes. SET1/MLL complexes are composed of a histone methyltransferase and the regulatory components WDR5, RbBP5, Ash2L, and DPY-30. MLL1/MLL2 complexes contain menin as additional component and are particularly important for the activation of HOX genes. We demonstrate that SENP3 is associated with MLL1/MLL2 complexes and catalyzes deSUMOylation of RbBP5. This is required for activation of a subset of HOX genes, including the developmental regulator DLX3. In the absence of SENP3, the association of menin and Ash2L with the DLX3 gene is impaired, leading to decreased H3K4 methylation and reduced recruitment of active RNA polymerase II. Importantly, the SENP3-DLX3 pathway dictates osteogenic differentiation of human stem cells, thus delineating the importance of balanced SUMOylation for epigenetic control of gene expression programs.

Mechanisms during Osteogenic Differentiation in Human Dental Follicle Cells.

Human dental follicle cells (DFCs) as periodontal progenitor cells are used for studies and research in regenerative medicine and not only in dentistry. Even if innovative regenerative therapies in medicine are often considered the main research area for dental stem cells, these cells are also very useful in basic research and here, for example, for the elucidation of molecular processes in the differentiation into mineralizing cells. This article summarizes the molecular mechanisms driving osteogenic differentiation of DFCs. The positive feedback loop of bone morphogenetic protein (BMP) 2 and homeobox protein DLX3 and a signaling pathway associated with protein kinase B (AKT) and protein kinase C (PKC) are presented and further insights related to other signaling pathways such as the WNT signaling pathway are explained. Subsequently, some works are presented that have investigated epigenetic modifications and non-coding ncRNAs and their connection with the osteogenic differentiation of DFCs. In addition, studies are presented that have shown the influence of extracellular matrix molecules or fundamental biological processes such as cellular senescence on osteogenic differentiation. The putative role of factors associated with inflammatory processes, such as interleukin 8, in osteogenic differentiation is also briefly discussed. This article summarizes the most important insights into the mechanisms of osteogenic differentiation in DFCs and is intended to be a small help in the direction of new research projects in this area.

Effects of Cellular Senescence on Dental Follicle Cells.

The dental follicle is part of the tooth germ, and isolated stem cells from this tissue (dental follicle cells; DFCs) are considered, for example, for regenerative medicine and immunotherapies. However somatic stem cells can also improve pharmaceutical research. Cell proliferation is limited by the induction of senescence, which, while reducing the therapeutic potential of DFCs for cell therapy, can also be used to study aging processes at the cellular level that can be used to test anti-aging pharmaceuticals. Unfortunately, very little is known about cellular senescence in DFCs. This review presents current knowledge about cellular senescence in DFCs.

p53 inhibits the osteogenic differentiation but does not induce senescence in human dental follicle cells.

Replicative senescence causes a reduced osteogenic differentiation potential of senescent dental follicle cells (DFCs). The transcription factor p53 is often involved in the induction of cellular senescence, but little is known about its role in DFCs. This study examined for the first time the role of p53 compared to its pro-proliferative antagonist E2F-1 in terms of osteogenic differentiation potential and induction of senescence. Protein expression of E2F-1 decreased during cell aging, while p53 was expressed constitutively. Gene silencing of E2F1 (E2F-1) inhibited the proliferation rate of DFCs and increased the induction of cellular senescence. The induction of cellular senescence is regulated independently of the gene expression of TP53 (p53), since its gene expression depends on the expression of E2F1. Moreover, gene silencing of TP53 induced E2F1 gene expression and increased cell proliferation, but did not affect the rate of induction of cellular senescence. TP53 knockdown further induced the alkaline phosphatase and mineralization in DFCs. However, the simultaneous silencing of TP53 and E2F1 did not inhibit the inductive effect of TP53 knockdown on osteogenic differentiation, indicating that this effect is independent of E2F-1. In summary, our results suggest that p53 inhibits osteogenic differentiation and cell proliferation in senescent DFCs, but is not significantly involved in senescence induction.

High endogenous expression of parathyroid hormone-related protein (PTHrP) supports osteogenic differentiation in human dental follicle cells.

Dental follicle cells (DFCs) are progenitor cells for mineralizing cells such as alveolar osteoblasts, but little is known about the mechanisms of the differentiation. Interestingly, different cell lines sometimes have different potentials to differentiate into mineralizing cells. In this study, we compared two different DFC lines, with one cell line (DFC_B) showing a high alkaline phosphatase (ALP) activity in long-term cultures with standard medium and a reliable mineralizing potential. However, the other cell line DFC_A shows low ALP activity in standard medium and almost no mineralization. Known osteogenic markers such as RUNX2 were similarly expressed in both cell lines. However, the proosteogenic signaling pathway of the bone morphogenetic protein (BMP) is induced in DFC_B, and the parathyroid hormone-related protein (PTHrP), which is involved in tooth root development, was also expressed more strongly. Previous studies have shown that the secreted PTHrP negatively regulate the transition from pre-osteoblastic progenitors to osteoblasts, but we showed that an inhibition of PTHrP gene expression reduced the ALP activity and the BMP-signaling pathway. In addition, endogenously expressed PTHrP is located in the cell nucleus. In contrast, supplementation of PTHrP or an inhibitor for the PTHrP receptor did not affect the ALP activity of DFC_B. In conclusion, our data suggest that a high endogenous expression of PTHrP in DFCs supports the induction of osteogenic differentiation via an intracrine mode.

WNT5A supports viability of senescent human dental follicle cells.

The osteogenic differentiation of dental follicle cells (DFCs) is inhibited by the onset of cellular senescence, but the cause for this is largely unknown. Recently it was shown that WNT5a, which is an inductor of the non-canonical WNT pathway, stimulates both cellular senescence and osteogenic differentiation of different cell types. In this study, we investigated the role of WNT5a for viability and osteogenic differentiation in human DFCs after the induction of cellular senescence. DFCs were cultivated until the induction of cellular senescence. The induction of cellular senescence was confirmed by ß-galactosidase staining, estimation of population doubling time, and slightly telomere length shortening. After induction of cellular senescence, the expression of WNT5A and the potential to induce the osteogenic differentiation decreased. Inhibition of WNT5A by specific siRNAs had significant effect on the viability of DFCs. Cell proliferation was reduced, whereas both cellular senescence and cell death were increased in DFCs. However, an inhibition of WNT5A did only slightly effect the osteogenic differentiation of DFCs. Our results suggest that WNT5A supports viability during both cell proliferation and osteogenic differentiation of DFCs.

The cell cycle regulator protein P16 and the cellular senescence of dental follicle cells.

Cellular senescence is a restricting factor for regenerative therapies with somatic stem cells. We showed previously that the onset of cellular senescence inhibits the osteogenic differentiation in stem cells of the dental follicle (DFCs), although the mechanism remains elusive. Two different pathways are involved in the induction of the cellular senescence, which are driven either by the cell cycle protein P21 or by the cell cycle protein P16. In this study, we investigated the expression of cell cycle proteins in DFCs after the induction of cellular senescence. The induction of cellular senescence was proved by an increased expression of ß-galactosidase and an increased population doubling time after a prolonged cell culture. Cellular senescence regulated the expression of cell cycle proteins. The expression of cell cycle protein P16 was up-regulated, which correlates with the induction of cellular senescence markers in DFCs. However, the expression of cyclin-dependent kinases (CDK)2 and 4 and the expression of the cell cycle protein P21 were successively decreased in DFCs. In conclusion, our data suggest that a P16-dependent pathway drives the induction of cellular senescence in DFCs.

The induction of cellular senescence in dental follicle cells inhibits the osteogenic differentiation.

Dental stem cells such as human dental follicle cells (DFCs) have opened new promising treatment alternatives for today's dental health issues such as periodontal tissue regeneration. However, cellular senescence represents a restricting factor to cultured stem cells, resulting in limited lifespan and reduced cell differentiation potential. Therefore, this study evaluated if and how DFCs exhibit features of cellular senescence after being expanded in cell culture. The cell proliferation of DFCs decreased, while the cell size increased during prolonged cell culture. Moreover, DFCs expressed the senescence-associated ß-galactosidase after a prolonged cell culture. The onset of senescence inhibited both the induction of osteoblast markers RUNX2 and osteopontin and the biomineralization of DFCs after stimulation of the osteogenic differentiation. In conclusion, we showed that a prolonged cell culture induces cellular senescence and inhibits the osteogenic differentiation in DFCs.

Genome-wide gene expression profiles of dental follicle stem cells.

OBJECTIVE: Dental stem cells (SCs) will be increasingly used for bone regeneration in the future. Recently, dental follicle cells (DFCs) from retained human third molars have been isolated and characterized as osteogenic progenitors. Although these results are promising for regenerative dentistry, molecular processes during osteogenic differentiation are not yet well understood. MATERIALS AND METHODS: This study compared DFCs before and during osteogenic differentiation. ALP activity was measured and cells were stained with alizarin red. Real-time RT-PCRs for osteogenic markers were done. The genome-wide expression profile was evaluated using a microarray. RESULTS: DFCs showed strong mineralization and increased expression of osteogenic marker genes during osteogenic differentiation. A microarray analysis showed regulated genes before and in the process of osteogenic differentiation (day 7). Several regulated genes in DFCs were associated with skeletal development. Bioinformatic analysis revealed a number of factors associated with dental follicle osteogenic differentiation. Osteogenic differentiation affected expression levels of the transcriptional regulators FOXC2 and ZNF219. CONCLUSION: In conclusion, the results yielded new objectives for further studies on transcription factors like FOXC2 or ETV1 and their role in dental SCs during osteogenic differentiation.

NOTCH1 signaling regulates the BMP2/DLX-3 directed osteogenic differentiation of dental follicle cells.

Dental follicle cells (DFCs) are dental stem/progenitor cells and the genuine precursors of alveolar osteoblasts and dental cementoblasts. A previous study showed that the transcription factor DLX3 (distal less homeobox 3) supports the osteogenic differentiation in DFCs via a positive feedback loop with the bone morghogenetic protein (BMP) 2. Until today, however, the control of this BMP2/DLX3 pathway by additional signaling pathways remains elusive. Previous studies also suggested that the NOTCH signaling pathway plays a role in the osteogenic differentiation of DFCs. In this study we showed that DLX3 overexpression and the initiation of the osteogenic differentiation by BMP2 or dexamethasone induced the NOTCH signaling pathway in DFCs. However, the induction of NOTCH-signaling impaired not only the osteogenic differentiation (ALP activity and mineralized nodules) but also the expression of the transcription factor DLX3 and the activation of the BMP-signaling pathway. So, NOTCH signaling plays a regulatory role for the osteogenic differentiation of DFCs. In conclusion, results of our study suggest that the NOTCH-signaling pathway, which is activated during the osteogenic differentiation of DFCs, regulates the BMP2/DLX3 directed differentiation of DFCs via a negative feed-back loop.

Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-ß1 and the activation of the FAK/ERK signaling pathway.

Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-ß1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-ß1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-ß1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-ß1 and the activation of the FAK/ERK signaling pathway.

Dexamethasone-related osteogenic differentiation of dental follicle cells depends on ZBTB16 but not Runx2.

Dental follicle cells (DFCs) can be artificially differentiated into mineralizing cells. With a dexamethasone-based differentiation protocol, transcription factors ZBTB16 and NR4A3 are highly upregulated but Runx2 and other osteogenic marker genes are not. Previous studies have suggested the involvement of a Runx2-independent differentiation pathway. The objective of this study is to further elucidate this mechanism. Differentiation of DFCs was examined by alkaline phosphatase (ALP) staining and ALP activity measurement, by Alizarin Red S staining and by real-time reverse transcription plus the polymerase chain reaction. ZBTB16 was overexpressed by using a transient transfection method. Resulting genome-wide gene expression changes were assessed by microarray. ZBTB16 and Runx2 were inhibited by short interfering RNA transfection. Promoter binding of ZBTB16 was evaluated by chromatin immunoprecipitation. Downregulation of Runx2 had no effect on dexamethasone-induced differentiation but was effective on BMP2-induced differentiation. Downregulation of ZBTB16, however, impaired dexamethasone-induced differentiation. Genes that were upregulated by dexamethasone induction were also upregulated by ZBTB16 overexpression. Genes that were not upregulated during dexamethasone-induced differentiation were also not regulated by ZBTB16 overexpression. ZBTB16 bound directly to the promoter regions of osterix and NR4A3 but not that of Runx2. Overexpression of ZBTB16 led to changes in the gene expression profile, whereby upregulated genes were overrepresented in osteogenesis-associated biological processes. Our findings suggest that, in DFCs, a Runx2-independent differentiation mechanism exists that is regulated by ZBTB16.

A site-specific phosphorylation of the focal adhesion kinase controls the formation of spheroid cell clusters.

Human dental follicle cells (DFCs) are ectomesenchymal multipotent stem cells that form spheroid cell clusters (SCCs) under serum free medium cell culture conditions (SFM). Until today, molecular mechanisms for the formation of SCCs are unknown. In this study a quantitative phosphoproteomics approach revealed regulated phosphorylated proteins in SCCs, which were derived from DFCs after 24 and 48 h in SFM. These regulated proteins were categorized using the Kyoto encyclopedia of genes and genomes program. Here, cellular processes and signaling pathway were identified such as the focal adhesion kinase (FAK) signaling pathway. In addition to the phosphoproteomics approach we showed that a specific phosphorylation of FAK (Y397) was required for the formation of SCCs. In conclusion, this study disclosed the phosphoproteome of SCCs for the first time and showed that the FAK signaling pathway is required for the formation of SCCs.

Butyrate stimulates the early process of the osteogenic differentiation but inhibits the biomineralization in dental follicle cells (DFCs).

Dental stem cells, especially dental follicle cells (DFCs) as precursor cells for the periodontium have interesting prospects for regenerative dentistry. During periodontitis, butyrate as a bacterial metabolite and inflammatory agent is often found in millimolar concentrations in periodontal pockets. This study evaluates the effects of butyrate on the proliferation and osteogenic differentiation of DFCs. We assessed cell viability/proliferation (BCA assay) and osteogenic differentiation (ALP activity, alizarin staining and RT PCR) of DFCs in vitro after butyrate supplementation. Butyrate concentrations of 20 mM or higher are toxic for DFCs. At a non-toxic concentration, butyrate promotes the expression of alkaline phosphatase and collagen type-1 but inhibits the formation of calcified nodules and the induction of RUNX2 and osteocalcin under osteogenic differentiation conditions. In conclusion, DFCs are resistant to physiological high concentrations of butyrate. Butyrate facilitates the osteogenic differentiation of DFCs in early stages but inhibits calcification at later stages of the differentiation process.

Transcription factors TP53 and SP1 and the osteogenic differentiation of dental stem cells.

Dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle cells (DFCs) have a promising potential for tissue engineering applications including periodontal and bone regeneration. However, little is known about the molecular mechanisms underlying osteogenic differentiation. In a previous study we detected that more than 35% of genes that are regulated during osteogenic differentiation of DFCs have promoter binding sites for the transcription factors TP53 and SP1. However, the role of these transcription factors in dental stem cells is still unknown. We hypothesize that both factors influence the processes of cell proliferation and differentiation in dental stem cells. Therefore, we transiently transfected DFCs and dental pulp stem cells (SHED; Stem cells from human exfoliated decidiuous teeth) with expression vectors for these transcription factors. After overexpression of SP1 and TP53, SP1 influenced cell proliferation and TP53 osteogenic differentiation in both dental cell types. The effects on cell proliferation and differentiation were less pronounced after siRNA mediated silencing of TP53 and SP1. This indicates that the effects we observed after TP53 and SP1 overexpression are indirect and subject of complex regulation. Interestingly, upregulated biological processes in DFCs after TP53-overexpression resemble the downregulated biological processes in SHED after SP1-overexpression. Here, regulated processes are involved in cell motility, wound healing and programmed cell death. In conclusion, our study demonstrates that SP1 and TP53 influence cell proliferation and differentiation and similar biological processes in both SHED and DFCs.

Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED).

Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness.

Dental stem cells for tooth regeneration: how far have we come and where next?

INTRODUCTION: Human dental stem cells are promising for tooth repair because of their differentiation potential. In 2018, this journal published a report on dental stem cell treatment options that had been attempted since the early 2000s. Although it is very difficult to follow every trend since then, new achievements have been made in the last 5 years. This review summarizes selected developments in dental stem cell research. AREAS COVERED: This article provides an overview of new developments with human dental stem cells and parts of these cells like extracellular vesicles for regenerative medicine. Preclinical research, clinical trials, and other works in the field of dental stem cells research for whole tooth engineering, dental pulp regeneration, periodontitis, and tooth root regeneration are summarized. In addition, works with dental stem cells for the regeneration of diseases that cannot be cured with the regeneration of dental tissues, such as diabetes, will be presented. EXPERT OPINION: Over the past five years, a number of studies using dental stem cells have improved new strategies for tooth repair. In addition, there are new dental stem cell products such as extracellular vesicles which, in combination with findings from basic research, will lead to new treatment options in the future.

Changes in AMPK activity induces cellular senescence in human dental follicle cells.

Dental Follicle Cells (DFCs) are somatic stem cells with a limited lifespan, but little is known about a possible mechanism of cellular senescence. Previous studies have shown that cellular senescence is associated with increased demand of glycolsis or the "glycolytic metabotype", which can be induced by activation of 5' adenosine monophosphate-activated protein kinase (AMPK), and decreased autophagy. This study examined the role of AMPK in inducing senescence in DFCs. During the induction of cellular senescence, AMPK activity was impaired, suggesting a negative impact on senescence induction. In line with this assumption, cellular senescence was induced upon inhibition of AMPK with a specific siRNA. In addition, after this inhibition, autophagy was also inhibited. Moreover, specific inhibition of autophagy promoted cellular senescence. However, inducers of AMPK such as metformin or AICAR surprisingly increased senescence in DFCs. Interestingly, autophagy was impaired after long-term induction of AMPK with AICAR and metformin. Moreover, activation of AMPK induces the consumption of glucose but decreases NAD/NADH ratio in DFCs that suggest not only "glycolytic metabotype" of DFCs but also Mitochondrial Dysfunction Associated Senescence (MiDAS). Both changes are highly associated with the induction of cellular senescence. Hence, both AMPK activation and inhibition promote the induction of cellular senecence of DFCs.