Periodontal Ligament-derived Stem Cells Research Articles

Photo biomodulation of dental derived stem cells to ameliorate regenerative capacity: In vitro study

BackgroundDental regeneration benefits from improving the features of dental derived stem cells. Gallium-aluminum-arsenide laser had a significant role in modification of cell behavior in different cell lines and culture conditions. Hence, exploring its mechanism and effect on dental derived stem cells would benefit prospective regenerative dental therapies. ObjectivesTo assess the impact of photo biomodulation by Low-Level-Laser on isolated Dental Pulp derived Stem Cells and Periodontal Ligament derived Stem Cells regarding their proliferation and osteogenic differentiation. MethodsIsolated DPSCs and PDLSCs from impacted third molars were subjected to Gallium-aluminum-arsenide laser for 12 sec and 3.6 J/cm2. The proliferative capacity was evaluated via 3-(4,5-dimethylthiazol-2-yl),2,5-diphenyltetrazolium bromide (MTT) Assay and Trypan blue stain. Cell osteogenic differentiation potentials were assessed by alkaline phosphatase assay and alizarin red stain, polymerase chain reaction was performed to quantify Nuclear factor Kappa gene expression. ResultsDPSCs subjected to laser bio-stimulation showed the best results regarding cell viability (MTT) and osteogenic differentiation (ALP assay), and calcium deposition at 3 intervals (3, 7, 14 days), meanwhile, PDLSCs subjected to laser bio-stimulation showed better result than control but less than DPSCs. While NF-KB gene expression was proven to be approximately comparable for both groups. Generally, the Photo-bio modulated groups showed better results than their control groups. ConclusionLow-level laser bio-stimulation (LLL) therapy improves DPSC and PDLSC osteogenic differentiation and proliferation via the activation of the NF-KB pathway. Also, the DPSCs outperformed PDLSCs in terms of performance. Clinical significanceThese results can be beneficial information and a reference database for more research in tissue engineering, dental therapy, and regeneration.

Therapeutic Potential of Oral-Derived Mesenchymal Stem Cells in Retinal Repair.

The retina has restricted regeneration ability to recover injured cell layer because of reduced production of neurotrophic factors and increased inhibitory molecules against axon regrowth. A diseased retina could be regenerated by repopulating the damaged tissue with functional cell sources like mesenchymal stem cells (MSCs). The cells are able to release neurotrophic factors (NFs) to boost axonal regeneration and cell maintenance. In the current study, we comprehensively explore the potential of various types of stem cells (SCs) from oral cavity as promising therapeutic options in retinal regeneration. The oral MSCs derived from cranial neural crest cells (CNCCs) which explains their broad neural differentiation potential and secret rich NFs. They are comprised of dental pulp SCs (DPSCs), SCs from exfoliated deciduous teeth (SHED), SCs from apical papilla (SCAP), periodontal ligament-derived SCs (PDLSCs), gingival MSCs (GMSCs), and dental follicle SCs (DFSCs). The Oral MSCs are becoming a promising source of cells for cell-free or cell-based therapeutic approach to recover degenerated retinal. These cells have various mechanisms of action in retinal regeneration including cell replacement and the paracrine effect. It was demonstrated that they have more neuroprotective and neurotrophic effects on retinal cells than immediate replacement of injured cells in retina. This could be the reason that their therapeutic effects would be weakened over time. It can be concluded that neuronal and retinal regeneration through these cells is most likely due to their NFs that dramatically suppress oxidative stress, inflammation, and apoptosis. Although, oral MSCs are attractive therapeutic options for retinal injuries, more preclinical and clinical investigations are required.

Senescent Markers Expressed by Periodontal Ligament-Derived Stem Cells (PDLSCs) Harvested from Patients with Periodontitis Can Be Rejuvenated by RG108.

Periodontal ligament (PDL) has become an elective source of mesenchymal stem cells (PDLSCs) in dentistry. This research aimed to compare healthy PDLSCs (hPDLSCs) and periodontitis PDLSCs (pPDLSCs) to ascertain any possible functional differences owing to their milieux of origin. Cells were tested in terms of colony-forming unit efficiency; multi differentiating capacity; immunophenotype, stemness, and senescent state were studied by flow cytometry, immunofluorescence, and β-galactosidase staining; gene expression using RT-PCR. Both hPDLSCs and pPDLSCs were comparable in terms of their immunophenotype and multilineage differentiation capabilities, but pPDLSCs showed a senescent phenotype more frequently. Thus, a selective small molecule inhibitor of DNA methyltransferase (DNMT), RG108, known for its effect on senescence, was used to possibly reverse this phenotype. RG108 did not affect the proliferation and apoptosis of PDLSCs, and it showed little effect on hPDLSCs, while a significant reduction of both p16 and p21 was detected along with an increase of SOX2 and OCT4 in pPDLSCs after treatment at 100 μM RG108. Moreover, the subset of PDLSCs co-expressing OCT4 and p21 decreased, and adipogenic potential increased in pPDLSCs after treatment. pPDLSCs displayed a senescent phenotype that could be reversed, opening new perspectives for the treatment of periodontitis.

Stem cell therapy based on human periodontal ligament stem cells preactivated with TNF-α as for human head and neck squamous cell carcinoma

ObjectiveMesenchymal stem cells (MSCs) express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following activation by various molecules and have antitumor effects against several cancer types. In particular, preactivation of MSCs with tumor necrosis factor-α (TNF-α) enhances TRAIL expression. We focused on the application of human periodontal ligament-derived stem cells (hPDLSCs) to stem cell-based cancer therapy, and evaluated their effects on head and neck squamous cell carcinoma (HNSCC) cells. MethodsTRAIL expression in hPDLSCs preactivated with TNF-α (A-hPDLSCs) was evaluated using real-time PCR, western blotting, and fluorescent immunocytochemistry. Subsequently, non-treated hPDLSCs (N-hPDLSCs) or A-hPDLSCs were cocultured with three types of HNSCC cells: HSC2, HSC3, and HSC4, and HNSCC cell apoptosis was assessed using flow cytometry. The migration ability of N-hPDLSCs and A-hPDLSCs toward HNSCC cells was also evaluated using a 3D gel invasion assay. In addition, conditioned medium (CM) derived from A-hPDLSCs was added to HNSCC cells, and cell proliferation ability was measured. ResultsA-hPDLSCs preactivated with 10 ng/mL TNF-α significantly upregulated the expression of TRAIL, increased apoptosis in HNSCC cells, and exhibited increased migration toward HNSCC cells compared to N-hPDLSCs. However, CM derived from A-hPDLSCs neither increased apoptosis nor suppressed HNSCC cell proliferation. ConclusionshPDLSCs are proposed to be beneficial for stem cell therapies for HNSCC. Upon TNF-α preactivation, hPDLSCs may migrate toward HNSCC cells and directly induce apoptosis.

In vitro investigation of canine periodontal ligament-derived mesenchymal stem cells: A possibility of promising tool for periodontal regeneration.

Recent investigations indicate that canine periodontal ligament-derived stem cells (cPDLSCs) may reveal a reliable strategy for repair of periodontal tissues via cell-based tissue engineering approaches. Due to limited research, this study aimed to demonstrate the phenotypic characterization of cPDLSc in comparison with canine bone marrow-derived mesenchymal stem cells (cBMSCs) in vitro. Mesenchymal stem cells (MSCs) were obtained from PDL and BM of five male adult Mongrel dogs. In vitro isolation and expansion as well as biologic characterization including colony unit formation (CFU), osteogenic and adipogenic differentiation, flow cytometric analysis of CD34 and CD44, and RT-PCR of alkaline phosphatase (ALP), osteocalcin (OCN), periostin (POSTN) and S100A4 were performed. Furthermore, electron microscopy analysis was done to complement the comparative research. CFU assay revealed that colonies of cPDLSCs presented 70% confluency with a more finite lifespan than BM-MSCs, showing a significant increase in cPDLSCs. Both types of MSCs showed osteogenic and adipogenic phenotypic characterized with clusters of mineralized depositions and lipid vacuoles, respectively. Both types of MSCs expressed CD44 with limited expression of CD34. RT-PCR of cPDLSCs revealed that expression of ALP, POSTN, OCN and S100A4 genes were significantly higher than those of BMSCs. In addition, comparison of SEM and revealed that cPDLSCs expressed more extracellular collagen fibers. The current study indicated that cPDLSCs show potency as a novel cellular therapy for periodontal regeneration a large animal model.

Three-Dimensional Culture of Cartilage Tissue on Nanogel-Cross-Linked Porous Freeze-Dried Gel Scaffold for Regenerative Cartilage Therapy: A Vibrational Spectroscopy Evaluation.

This study presents a set of vibrational characterizations on a nanogel-cross-linked porous freeze-dried gel (NanoCliP-FD gel) scaffold for tissue engineering and regenerative therapy. This scaffold is designed for the in vitro culture of high-quality cartilage tissue to be then transplanted in vivo to enable recovery from congenital malformations in the maxillofacial area or crippling jaw disease. The three-dimensional scaffold for in-plate culture is designed with interface chemistry capable of stimulating cartilage formation and maintaining its structure through counteracting the dedifferentiation of mesenchymal stem cells (MSCs) during the formation of cartilage tissue. The developed interface chemistry enabled high efficiency in both growth rate and tissue quality, thus satisfying the requirements of large volumes, high matrix quality, and superior mechanical properties needed in cartilage transplants. We characterized the cartilage tissue in vitro grown on a NanoCliP-FD gel scaffold by human periodontal ligament-derived stem cells (a type of MSC) with cartilage grown by the same cells and under the same conditions on a conventional (porous) atelocollagen scaffold. The cartilage tissues produced by the MSCs on different scaffolds were comparatively evaluated by immunohistochemical and spectroscopic analyses. Cartilage differentiation occurred at a higher rate when MSCs were cultured on the NanoCliP-FD gel scaffold compared to the atelocollagen scaffold, and produced a tissue richer in cartilage matrix. In situ spectroscopic analyses revealed the cell/scaffold interactive mechanisms by which the NanoCliP-FD gel scaffold stimulated such increased efficiency in cartilage matrix formation. In addition to demonstrating the high potential of human periodontal ligament-derived stem cell cultures on NanoCliP-FD gel scaffolds in regenerative cartilage therapy, the present study also highlights the novelty of Raman spectroscopy as a non-destructive method for the concurrent evaluation of matrix quality and cell metabolic response. In situ Raman analyses on living cells unveiled for the first time the underlying physiological mechanisms behind such improved chondrocyte performance.

Chondroprotective Effects of Periodontal Ligament-Derived Stem Cells Conditioned Medium on Articular Cartilage After Impact Injury.

Paracrine factors secreted in the conditioned media (CMs) of periodontal ligament-derived stem cells (PDLSCs) have been shown to downregulate inflammatory effects of interleukin (IL)-1β on chondrocytes wherein milk fat globule-epidermal growth factor 8 (MFG-E8) is one of the PDLSCs' highly secretory proteins. Therefore, the objective of this study was to investigate the ability of PDLSC CMs and MFG-E8 to reduce the inflammatory effects of impact injury on porcine talar articular cartilage (AC) and IL-1β on chondrocytes, respectively. Stem cells were isolated from human periodontal ligaments. The MFG-E8 content in CM collected at 5% and 20% oxygen was measured by ELISA assay and compared across subcultures and donors. AC samples were divided into three groups: control, impact, and impact+CM. Chondrocytes were isolated from pig knees and were divided into three groups: control, IL-1β, and IL-1β+MFG-E8. Gene expression data were analyzed by reverse transcription-polymerase chain reaction. It was found that impact load and IL-1β treatment upregulated IL-1β, TNF-α, ADAMTS-4, and ADAMTS-5 gene expression in AC and chondrocytes, respectively. PDLSCs-CM prevented the upregulation of all four genes due to impact, whereas MFG-E8 prevented upregulation of IL-1β, ADAMTS-4, and ADAMTS-5 in chondrocytes, but it did not prevent TNF-α upregulation. There were no significant differences in MFG-E8 content in CM among oxygen levels, passage numbers, or donors. The findings suggested that MFG-E8 is an effective anti-inflammatory agent contributing to the chondroprotective effects of PDLSCs-CM on acutely injured AC. Thus, introducing PDLSCs-CM to sites of acute traumatic AC injury could prevent the development of post-traumatic osteoarthritis.

Metabolic Reconfiguration Activates Stemness and Immunomodulation of PDLSCs.

Periodontal ligament derived stem cells (PDLSC) are adult multipotent mesenchymal-like stem cells (MSCs) that can induce a promising immunomodulation to interact with immune cells for disease treatment. Metabolic reconfiguration has been shown to be involved in the immunomodulatory activity of MSCs. However, the underlying mechanisms are largely unknown, and it remains a challenging to establish a therapeutic avenue to enhance immunomodulation of endogenous stem cells for disease management. In the present study, RNA-sequencing (RNA-seq) analysis explores that curcumin significantly promotes PDLSC function through activation of MSC-related markers and metabolic pathways. In vitro stem cell characterization further confirms that self-renewal and multipotent differentiation capabilities are largely elevated in curcumin treated PDLSCs. Mechanistically, RNA-seq reveals that curcumin activates ERK and mTOR cascades through upregulating growth factor pathways for metabolic reconfiguration toward glycolysis. Interestingly, PDLSCs immunomodulation is significantly increased after curcumin treatment through activation of prostaglandin E2-Indoleamine 2,3 dioxygenase (PGE2-IDO) signaling, whereas inhibition of glycolysis activity by 2-deoxyglucose (2-DG) largely blocked immunomodulatory capacity of PDLSCs. Taken together, this study provides a novel pharmacological approach to activate endogenous stem cells through metabolic reprogramming for immunomodulation and tissue regeneration.

Near Infrared Laser Photobiomodulation of Periodontal Ligament Stem Cells.

To determine the effect of different energy densities of near infrared diode lasers with wavelengths of 810 or 940 nm on the proliferation and survival of periodontal ligament derived stem cells (PDLSCs). After isolation and characterisation, PDLSCs were cultured in clear 96-well plates. Each well was irradiated by either 810 nm (L1) or 940 nm (L2) lasers, with energy densities of 0.5, 1.5 and 2.5 J/cm2 and an output power of 100 mW. A non-irradiated well was used as a control. Cellular viability was measured 24 hours after irradiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and proliferation was measured 24, 48 and 72 hours after irradiation using trypan blue staining and counting. Propidium iodide (PI) staining was used to identify any pyknotic nuclei or nuclear fragmentation 72 hours after irradiation. An increase in viability was observed only in the group with the 940 nm laser irradiation at energy density of 2.5 J/cm2 (P < 0.001). The proliferation of cells was significantly increased in the group with 940 nm laser irradiation at energy density of 2.5 J/cm2 at all the time points examined in comparison to other groups (P < 0.001). PI staining showed no change in cell nuclei in any of the groups. Irradiation of PDLSCs with a 940 nm laser at an energy density of 2.5 J/cm2 could promote efficient cell proliferation.

Comparison of Cellular and Differentiation Characteristics of Mesenchymal Stem Cells Derived from Human Gingiva and Periodontal Ligament.

ABSTRACTObjectives:Dental tissues possess multipotent stem cells with varying biological properties. The present study was aimed to establish a primary culture of human gingiva-derived mesenchymal stem cells (GMSCs) and periodontal ligament-derived stem cells (PDLSCs) from periodontally healthy subjects and compare their biological characteristics.Materials and Methods:Gingival and periodontal ligament (PDL) tissues were collected from extracted premolar teeth of five healthy subjects and primary cultures were established. Basic biological characteristics, such as cell morphology, viability, proliferation capacity, and colony-forming units, and in vitro osteogenic and adipogenic differentiation potential were performed at passage 3 of GMSCs and PDLSCs. This was followed by immuno-phenotyping and flow cytometric analysis for identification of positive mesenchymal stem cell (MSC) markers, such as CD73, CD90, and CD105, and negative markers CD45 and CD34.Statistical Analysis Used:One-way analysis of variance (ANOVA).Results:Primary cultures of GMSCs and PDLSCs were successfully established. Cells exhibited a fibroblast-like morphology with a homogeneous population at passage 3. Cells derived from both tissues were highly viable (>95%), proliferative, and capable of forming colonies. Both cells did not exhibit any noticeable differences in cellular properties. Immunofluorescence and flow cytometric analyses showed positivity for MSC markers, CD73, CD90, and CD105, and negativity for CD34 and CD45. Furthermore, GMSCs and PDLSCs were capable of differentiating in vitro into osteocytes as evidenced by Alizarin red-S staining, and adipocytes as demonstrated by oil red O staining.Conclusions:The results of the present study indicate that both GMSCs and PDLSCs have similar cellular characteristics and mesenchymal differentiation potential. Therefore, they may serve as an equally potent source of stem cells for use in cell-based periodontal therapies.

Activation of Functional Somatic Stem Cells Promotes Endogenous Tissue Regeneration

Periodontal ligament derived stem cells (PDLSCs) are capable of differentiating into multiple cell types and inducing a promising immunomodulation for tissue regeneration and disease treatment. However, it is still challenging to develop a practical approach to activate endogenous stem cells for tissue self-healing and regeneration. In this study, transcriptome analysis reveals that resveratrol promotes PDLSC stemness through activation of stem cell, osteoprogenitor, and chondroprogenitor markers. Self-renewal and multipotent differentiation abilities are also improved in resveratrol-treated PDLSCs. In addition, immunomodulation of PDLSCs is dramatically increased after resveratrol treatment. Mechanistically, we show that resveratrol activates ERK/WNT crosstalk through elevation of olfactory and growth factor signaling pathways to upregulate the expression levels of RUNX2 and FASL for osteogenesis and immunomodulation, respectively. By using a periodontitis animal model, administration of resveratrol partially rescues bone loss through activation of endogenous somatic stem cells and inhibition of inflammatory T-cell infiltration. Taken together, our findings identify a novel pharmacological approach to achieve autotherapies for endogenous tissue regeneration.

Differentiation of human adult-derived stem cells towards a neural lineage involves a dedifferentiation event prior to differentiation to neural phenotypes

Although it has been reported that mesenchymal stem cells isolated from adult tissues can be induced to overcome their mesenchymal fate and transdifferentiate into neural cells, the findings and their interpretation have been challenged. The main argument against this process is that the cells rapidly adopt neuron-like morphologies through retraction of the cytoplasm rather than active neurite extension. In this study, we examined the sequence of biological events during neural differentiation of human periodontal ligament-derived stem cells (hPDLSCs), human bone marrow-derived stem cells (hBMSCs) and human dental pulp-derived stem cells (hDPSCs) by time-lapse microscopy. We have demonstrated that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron-like cells without passing through a mitotic stage and that they shrink dramatically and change their morphology to that of neuron-like cells through active neurite extension. Furthermore, we observed micronuclei movement and transient cell nuclei lobulation concurrent to in vitro neurogenesis from hBMSCs and hDPSCs. Our results demonstrate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step followed by differentiation to neural phenotypes, and therefore we definitively confirm that the rapid acquisition of the neural phenotype is via a differentiation trait.

Anti-Inflammatory Effects of Conditioned Medium of Periodontal Ligament-Derived Stem Cells on Chondrocytes, Synoviocytes, and Meniscus Cells.

Osteoarthritis (OA) is the most common type of arthritis, afflicting millions of people in the world. Elevation of inflammatory mediators and enzymatic matrix destruction is often associated with OA. Therefore, the objective of this study was to investigate the effects of conditioned medium from periodontal ligament-derived stem cells (PDLSCs) on inflammatory and catabolic gene expressions of chondrocytes, synoviocytes, and meniscus cells under in vitro inflammatory condition. Stem cells were isolated from human periodontal ligaments. Conditioned medium was collected and concentrated 20 × . Chondrocytes, synoviocytes, and meniscus cells were isolated from pig knees and divided into four experimental groups: serum-free media, serum-free media+interleukin-1β (IL-1β) (10 ng/mL), conditioned media (CM), and CM+IL-1β. Protein content and extracellular vesicle (EV) miRNAs of CM were analyzed by liquid chromatography-tandem mass spectrometry and RNA sequencing, respectively. It was found that the IL-1β treatment upregulated the expression of IL-1β, tumor necrosis factor-α (TNF-α), MMP-13, and ADAMTS-4 genes in the three cell types, whereas PDLSC-conditioned medium prevented the upregulation of gene expression by IL-1β in all three cell types. This study also found that there was consistency in anti-inflammatory effects of PDLSC CM across donors and cell subcultures, while PDLSCs released several anti-inflammatory factors and EV miRNAs at high levels. OA has been suggested as an inflammatory disease in which all intrasynovial tissues are involved. PDLSC-conditioned medium is a cocktail of trophic factors and EV miRNAs that could mediate different inflammatory processes in various tissues in the joint. Introducing PDLSC-conditioned medium to osteoarthritic joints could be a potential treatment to prevent OA progression by inhibiting inflammation.

Stem cell therapies for periodontal tissue regeneration: a network meta-analysis of preclinical studies

BackgroundPeriodontal tissue regeneration (PTR) is the ultimate goal of periodontal therapy. Currently, stem cell therapy is considered a promising strategy for achieving PTR. However, there is still no conclusive comparison that distinguishes clear hierarchies among different kinds of stem cells.MethodsA systematic review and network meta-analysis (NMA) was performed using MEDLINE (via PubMed), EMBASE, and Web of Science up to February 2020. Preclinical studies assessing five types of stem cells for PTR were included; the five types of stem cells included periodontal ligament-derived stem cells (PDLSCs), bone marrow-derived stem cells (BMSCs), adipose tissue-derived stem cells (ADSCs), dental pulp-derived stem cells (DPSCs), and gingival-derived stem cells (GMSCs). The primary outcomes were three histological indicators with continuous variables: newly formed alveolar bone (NB), newly formed cementum (NC), and newly formed periodontal ligament (NPDL). We performed pairwise meta-analyses using a random-effects model and then performed a random-effects NMA using a multivariate meta-analysis model.ResultsSixty preclinical studies assessing five different stem cell-based therapies were identified. The NMA showed that in terms of NB, PDLSCs (standardized mean difference 1.87, 95% credible interval 1.24 to 2.51), BMSCs (1.88, 1.17 to 2.59), and DPSCs (1.69, 0.64 to 2.75) were statistically more efficacious than cell carriers (CCs). In addition, PDLSCs were superior to GMSCs (1.49, 0.04 to 2.94). For NC, PDLSCs (2.18, 1.48 to 2.87), BMSCs (2.11, 1.28 to 2.94), and ADSCs (1.55, 0.18 to 2.91) were superior to CCs. For NPDL, PDLSCs (1.69, 0.92 to 2.47) and BMSCs (1.41, 0.56 to 2.26) were more efficacious than CCs, and PDLSCs (1.26, 0.11 to 2.42) were superior to GMSCs. The results of treatment hierarchies also demonstrated that the two highest-ranked interventions were PDLSCs and BMSCs.ConclusionPDLSCs and BMSCs were the most effective and well-documented stem cells for PTR among the five kinds of stem cells evaluated in this study, and there was no statistical significance between them. To translate the stem cell therapies for PTR successfully in the clinic, future studies should utilize robust experimental designs and reports.

Attenuated regenerative properties in human periodontal ligament-derived stem cells of older donor ages with shorter telomere length and lower SSEA4 expression.

Periodontal ligament (PDL) stem cell properties are critical in the periodontal tissue regeneration for periodontitis. Previously, we have demonstrated that cigarette smoking attenuates PDL-derived stem cell (PDLSC) regenerative properties. Here, we report the findings on the regenerative properties of human PDLSCs with different donor ages and the underlying mechanisms. Human PDLSCs from 18 independent donors were divided into different age groups (≤ 20, 20-40, and > 40years old). The proliferation of PDLSCs with donor age of ≤ 20years old was significantly higher than that of the 20-40- and > 40-years-old groups, whereas the migration of PDLSCs with donor age of ≤ 20 and 20-40years old was significantly higher than that of the > 40-years-old group. Moreover, the mesodermal lineage differentiation capabilities of PDLSCs were also higher in the donor age group of ≤ 20years old than the donor age of > 40years old. In addition, shorter telomere length and lower expression of SSEA4 were found in PDLSCs with donor age of > 40years old, compared with those with donor age of ≤ 20-years-old group. Besides, PDLSCs with donor age of 20-40 and > 40years old had higher IL6 and CXCL8 gene expressions. In summary, results from this study revealed the attenuated proliferation, migration, and mesodermal lineage differentiation properties in human PDLSCs with older donor ages. Donor age of PDLSCs should be considered as the selection criteria for the periodontal tissue regeneration treatment.

Non-Thermal Bio-Compatible Plasma Induces Osteogenic Differentiation of Human Mesenchymal Stem/Stromal Cells With ROS-Induced Activation of MAPK

The application of non-thermal bio-compatible plasma (NBP) for stem cell differentiation is promising in tissue engineering. However, the differentiation efficiency of NBP treatment on various types of human tissue-derived stem cells and the underlying mechanisms is yet understood. This study is the first time to investigate the role of NBP in inducing differentiation and its potential molecular mechanism by using human bone marrow-derived stem cells (hBMSCs) and human periodontal ligament-derived stem cells (hPDLSCs). Our results showed that NBP promote osteogenic differentiation of hPDLSCs more effectively than hBMSCs under the same treatment condition, indicating a tissue-dependent manner of NBP interacts with stem cells. Furthermore, an increase of intracellular reactive oxygen species (ROS) production of hPDLSCs and antioxidant enzymes activation was observed after NBP treatment. Particularly, mitogen-activated protein kinases (MAPKs) level was also increased and in consistent with ROS level increase. Taken together, this study revealed that with NBP induction, hPDLSCs is a more suitable stem cell source than hBMSCs for bone regeneration and tissue engineering, and ROS-induced activation of MAPKs are possibly involved in the osteogenic differentiation process.

MicroRNA-132 directs human periodontal ligament-derived neural crest stem cell neural differentiation.

Neurogenesis is the basis of stem cell tissue engineering and regenerative medicine for central nervous system (CNS) disorders. We have established differentiation protocols to direct human periodontal ligament-derived stem cells (PDLSCs) into neuronal lineage, and we recently isolated the neural crest subpopulation from PDLSCs, which are pluripotent in nature. Here, we report the neural differentiation potential of these periodontal ligament-derived neural crest stem cells (NCSCs) as well as its microRNA (miRNA) regulatory mechanism and function in NCSC neural differentiation. NCSCs, treated with basic fibroblast growth factor and epidermal growth factor-based differentiation medium for 24days, expressed neuronal and glial markers (βIII-tubulin, neurofilament, NeuN, neuron-specific enolase, GFAP, and S100) and exhibited glutamate-induced calcium responses. The global miRNA expression profiling identified 60 upregulated and 19 downregulated human miRNAs after neural differentiation, and the gene ontology analysis of the miRNA target genes confirmed the neuronal differentiation-related biological functions. In addition, overexpression of miR-132 in NCSCs promoted the expression of neuronal markers and downregulated ZEB2 protein expression. Our results suggested that the pluripotent NCSCs from human periodontal ligament can be directed into neural lineage, which demonstrate its potential in tissue engineering and regenerative medicine for CNS disorders.

Notch Signaling Activation Enhances Human Adipose-Derived Stem Cell Retinal Differentiation.

Retinal disease treatment by stem cell-based replacement relies on stem cell differentiation into retinal cells. We previously demonstrated that human periodontal ligament-derived stem cells can be directed into retinal lineage upon induction. Here, we report the transdifferentiation potential of human adipose-derived stem cells (ASCs) into retinal lineage and its enhancement by Notch signaling modulation. Human ASCs, isolated from abdominal fat, expressed mesenchymal but not hematopoietic stem cell markers, and they can differentiate into adipocytes, chondrocytes, and osteoblasts in vitro. Upon noggin/Dkk-1/IGF-1 induction, the treated ASCs showed elevated expression of retinal progenitor, retinal ganglion, and photoreceptor cell markers as well as the glutamate-evoked calcium response, which was not observed in the noninduced cells. Compared to the regular induction treatment, Notch signaling activation by JAG1 enhanced the expression of retinal progenitor and precursor markers without affecting the glutamate-evoked calcium response. In contrast, Notch signaling inhibition by DAPT showed more retinal ganglion cells, but delayed the response to glutamate stimulation. In summary, our results revealed that human ASCs possess a retinal transdifferentiation potential upon noggin/Dkk-1/IGF-1 induction, which can further be enhanced by Notch signaling activation.

MicroRNA-543 functions as an osteogenesis promoter in human periodontal ligament-derived stem cells by inhibiting transducer of ERBB2, 2.

Previous research has indicated that altered expression of microRNAs (miRNAs) is in connection with osteogenesis of human periodontal ligament-derived stem cells (hPDLSCs). We investigated the mechanisms by which miR-543 promotes osteogenic differentiation of hPDLSCs. First, the expression of miR-543 in hPDLSCs cultured with or without an osteogenic inductive cocktail was explored. Then, the function of miR-543 during osteogenesis of hPDLSCs was investigated by overexpressing and inhibiting miR-543. Next, 3 databases were used to predict target genes of miR-543 and a luciferase report was used to validate the direct regulation of miR-543 on the target gene. Further, a rescue experiment using co-transfection of miR-543 mimic and target mimic was performed to evaluate whether overexpressing the target gene could partly rescue the efficiency of overexpressing miR-543 on osteogenesis in hPDLSCs. miR-543 was upregulated during osteogenic differentiation of hPDLSCs. Functional experiments showed that overexpressing miR-543 could enhance osteogenesis, while inhibiting miR-543 resulted in reduced formation of mineralized nodules. The transducer of ERBB2, 2 (TOB2) was identified as a target gene of miR-543 and luciferase report revealed that miR-543 interacts directly with the 3'-untranslated repeat sequence of TOB2 mRNA. Overexpression of miR-543 inhibited the expression of TOB2 in both mRNA and protein levels while inhibiting miR-543 increased. Furthermore, the rescue experiment confirmed the promotional role of miR-543 TOB2 expression could be abrogated by overexpressing TOB2, which also had the effect of reducing osteogenic differentiation. Our research confirmed that miR-543 is a promoter of osteogenesis in hPDLSCs, acting by inhibiting its target gene TOB2, which suggests that miR-543 may be a potential therapy for bone loss in periodontitis.

Human Periodontal Ligament-Derived Stem Cells Promote Retinal Ganglion Cell Survival and Axon Regeneration After Optic Nerve Injury.

Optic neuropathies are the leading cause of irreversible blindness and visual impairment in the developed countries, affecting more than 80 million people worldwide. While most optic neuropathies have no effective treatment, there is intensive research on retinal ganglion cell (RGC) protection and axon regeneration. We previously demonstrated potential of human periodontal ligament-derived stem cells (PDLSCs) for retinal cell replacement. Here, we report the neuroprotective effect of human PDLSCs to ameliorate RGC degeneration and promote axonal regeneration after optic nerve crush (ONC) injury. Human PDLSCs were intravitreally injected into the vitreous chamber of adult Fischer rats after ONC in vivo as well as cocultured with retinal explants in vitro. Human PDLSCs survived in the vitreous chamber and were maintained on the RGC layer even at 3 weeks after ONC. Immunofluorescence analysis of βIII-tubulin and Gap43 showed that the numbers of surviving RGCs and regenerating axons were significantly increased in the rats with human PDLSC transplantation. In vitro coculture experiments confirmed that PDLSCs enhanced RGC survival and neurite regeneration in retinal explants without inducing inflammatory responses. Direct cell-cell interaction and elevated brain-derived neurotrophic factor secretion, but not promoting endogenous progenitor cell regeneration, were the RGC protective mechanisms of human PDLSCs. In summary, our results revealed the neuroprotective role of human PDLSCs by strongly promoting RGC survival and axonal regeneration both in vivo and in vitro, indicating a therapeutic potential for RGC protection against optic neuropathies. Stem Cells 2018;36:844-855.