Functional differences in mesenchymal stromal cells from human dental pulp and periodontal ligament

Clinically reported reparative benefits of mesenchymal stromal cells (MSCs) are majorly attributed to strong immune‐modulatory abilities not exactly shared by fibroblasts. However, MSCs remain heterogeneous populations, with unique tissue‐specific subsets, and lack of clear‐cut assays defining therapeutic stromal subsets adds further ambiguity to the field. In this context, in‐depth evaluation of cellular characteristics of MSCs from proximal oro‐facial tissues: dental pulp (DPSCs) and periodontal ligament (PDLSCs) from identical donors provides an opportunity to evaluate exclusive niche‐specific influences on multipotency and immune‐modulation. Exhaustive cell surface profiling of DPSCs and PDLSCs indicated key differences in expression of mesenchymal (CD105) and pluripotent/multipotent stem cell–associated cell surface antigens: SSEA4, CD117, CD123 and CD29. DPSCs and PDLSCs exhibited strong chondrogenic potential, but only DPSCs exhibited adipogenic and osteogenic propensities. PDLSCs expressed immuno‐stimulatory/immune‐adhesive ligands like HLA‐DR and CD50, upon priming with IFNγ, unlike DPSCs, indicating differential response patterns to pro‐inflammatory cytokines. Both DPSCs and PDLSCs were hypo‐immunogenic and did not elicit robust allogeneic responses despite exposure to IFNγ or TNFα. Interestingly, only DPSCs attenuated mitogen‐induced lympho‐proliferative responses and priming with either IFNγ or TNFα enhanced immuno‐modulation capacity. In contrast, primed or unprimed PDLSCs lacked the ability to suppress polyclonal T cell blast responses. This study indicates that stromal cells from even topographically related tissues do not necessarily share identical MSC properties and emphasizes the need for a thorough functional testing of MSCs from diverse sources with respect to multipotency, immune parameters and response to pro‐inflammatory cytokines before translational usage.     

Rho‐kinase inhibitor Y‐27632 facilitates the proliferation,migration and pluripotency of human periodontal ligament stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell‐based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self‐renewal, lineage commitment and apoptosis. Trans‐4‐[(1R)‐aminoethyl]‐N‐(4‐pyridinyl)cylohexanecarboxamidedihydrochloride (Y‐27632), Rho‐associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti‐apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit‐8 (CCK8), 5‐ethynyl‐2′‐deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound‐healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to determine the effects of Y‐27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y‐27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y‐27632 induced PDLSCs proliferation through extracellular‐signal‐regulated kinase (ERK) signalling cascade. Therefore, control of Rho‐kinase activity may enhance the efficiency of stem cell‐based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.     

The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells

Platelet-rich plasma (PRP) is an emerging therapeutic application because PRP contains various growth factors that have beneficial effects on tissue regeneration and engineering. Mesenchymal stem cells and PRP derived from peripheral blood have been well studied. In this study, we investigated the effects of PRP derived from human umbilical cord blood (UCB-PRP) on proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs). Three types of dental stem cells were primarily isolated and characterized by flow cytometric analysis. Dental stem cells were exposed to various concentrations of UCB-PRP, which resulted in the proliferation of dental stem cells. Treatment with 2% UCB-PRP resulted in the highest level of proliferation. The ALP activity of DPSCs and PDLSCs increased following treatment with UCB-PRP in a dose-dependent manner up to a concentration of 2%. ALP activity decreased with higher concentration of UCB-PRP. The effects of UCB-PRP on calcium deposition were similar to those on proliferation and ALP activity. Treatment with 2% UCB-PRP resulted in the highest calcium depositions in DPSCs and PDLSCs; however, treatment with 1% UCB-PRP resulted in the highest calcium deposition in SHEDs. The concentrations of platelet-derived growth factor-AB and transforming growth factor-β1 in UCB-PRP were investigated and found to be comparable to the amounts in peripheral blood. Overall, UCB-PRP had beneficial effects on the proliferation and osteogenic differentiation of dental stem cells. Determination of the optimal concentration of UCB-PRP requires further investigation for clinical applications.     

Proteome of Human Stem Cells from Periodontal Ligament and Dental Pulp

Background

Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro.

Methodology/Principal Findings

The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4–7 and 6–9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin.

Conclusion/Significance

This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.     

Vitamin C alleviates the senescence of periodontal ligament stem cells through inhibition of Notch3 during long‐term culture

Periodontal ligament stem cells (PDLSCs), as potential “seed cells” for periodontal tissue repair and regeneration, require to be expanded in vitro for a large scale. Senescence of PDLSCs occurred during long‐term culture may compromise the therapeutic effects of PDLSCs. Medium supplements may be useful in antisenescence. However, the effects and mechanisms of vitamin C (Vc) treatment on PDLSCs during long‐term culture are still unclear. In this study, we identified that Vc‐treated PDLSCs cells maintained a slender morphology, higher growth rate and migration capacity, stemness, and osteogenic differentiation capability during a long‐term culture. Moreover, we also identified that Notch3 was significantly upregulated during the cell senescence, and Vc treatment alleviated the senescence of PDLSCs through inhibition of Notch3 during long‐term culture. In summary, Vc treatment suppressed PDLSCs senescence by reducing the expression of Notch3 and might be a simple and useful strategy to inhibit cellular senescence during the cell long‐term culture.     

Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Background information. Although adult bone‐marrow‐derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non‐dental stem‐cell‐mediated odontogenesis remain an open question. Results. STRO‐1⁺ (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic‐activated cell‐sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2‐day‐old rat incisors. Co‐cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up‐regulated alkaline phosphatase activity, cell‐cycle modification, and the expression of tooth‐specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth‐shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin—pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin‐like structures and fibrous connective tissues respectively. Conclusions. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non‐dental stem cell populations.     

Development of a serum‐free system to expand dental‐derived stem cells: PDLSCs and SHEDs

Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. In this study, we investigated the development of a chemically defined, serum‐free media (K‐M) for the expansion of human periodontal ligament stem cells (PDLSCs) and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were performed comparing cells in serum‐containing media (FBS‐M) with cells cultured in four different serum‐free medium and these demonstrated that in these medium, the cell proliferation of both cell types was significantly less than the proliferation of cells in FBS‐M. Additional proliferation assays were performed using pre‐coated fibronectin (FN) tissue culture plates and of the four serum‐free medium, only K‐M enabled PDLSCs and SHEDs to proliferate at higher rates than cells cultured in FBS‐M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs exhibited similar osteogenic potential whether cultured in K‐M or FBS‐M, and, additionally, cells retained their multipotency in K‐M as seen by expression of chondrogenic and adipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K‐M or FBS‐M. Taken together, the data suggest that K‐M can support the expansion of PDLSCs and SHEDs and maintenance of their multipotency. J. Cell. Physiol. 226: 66–73, 2010. © 2010 Wiley‐Liss, Inc.     

牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。     

Metformin facilitates the proliferation,migration, and osteogenic differentiation of periodontal ligament stem cells in vitro

Periodontitis is one of the main causes of tooth loss and has been confirmed as the sixth complication of diabetes. Metformin promotes the osteogenic differentiation of stem cells. Periodontal ligament stem cells (PDLSCs) are the best candidate stem cells for periodontal tissue regeneration. Herein, we aimed to identify the effects of metformin on the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. PDLSCs were isolated by limiting dilution, and their characteristics were assessed by colony formation assay and flow cytometry. Cell counting and migration assays were used to investigate the effects of metformin on proliferation and migration. The osteogenic differentiation ability of PDLSCs was detected by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene and protein levels of osteogenesis‐related markers were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis, respectively. Metformin treatment at 10 μM did not affect PDLSC proliferation, while at 50 and 100 μM, metformin time‐dependently enhanced PDLSC proliferation and significantly increased cell numbers after 5 and 7 days of stimulation (P < 0.05). In addition, 50 μM metformin exhibited a maximal effect on migration, ALP activity, and mineral deposition (P < 0.05). Furthermore, 50 μM metformin significantly upregulated the gene expression levels of ALP, BSP, OPN, OCN, and Runx2 and the protein expression of ALP and Runx2 (P < 0.05). In summary, our study confirms that metformin facilitates the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro and could be used as a new strategy for periodontal tissue regeneration.     

Characterisation of human dental stem cells and buccal mucosa fibroblasts

Human craniofacial stem cells are recently discovered sources of putative mesenchymal stem cells that hold great promise for autogenic or allogenic cell therapy and tissue engineering. Prior to employing these cells in clinical applications, they must be thoroughly investigated and characterized. In this study, the surface marker expression was investigated on dental pulp stem cells (DPSCs), dental follicle cells (DFCs), periodontal ligament stem cells (PDLSCs), and buccal mucosa fibroblasts (BMFs) utilising surface markers for flow cytometry. The osteogenic potential was also examined by bone-associated markers alkaline phosphatase, Runx2, collagen type I, osteocalcin, and osteopontin. The results from our study demonstrate that the dental cell sources exhibit comparable surface marker and bone-associated marker profiles parallel to those of other mesenchymal stem cell sources, yet distinct from the buccal mucosa fibroblasts. Our data support evidence towards clinical applicability of dental stem cells in hard tissue regeneration.     

Comparative proteomics analysis of human stem cells from dental gingival and periodontal ligament

Dental stem cells isolated from oral tissues have been shown to provide with high proliferation ability and multilineage differentiation potential. Gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs), kinds of dental stem cells, can be used as substitutes for tissue repair materials because of their similar regenerative functions. In this study, we aim to explore the similarities and differences between the protein profiles of GMSCs and PDLSCs through quantitative proteomics. A total of 2821 proteins were identified and retrieved, of which 271 were upregulated and 57 were downregulated in GMSCs compared to PDLSCs. Gene Ontology (GO) analysis demonstrated that the 328 differentially abundant proteins (DAPs) were involved in the regulation of gene expression, metabolism, and signal transduction in biological process, mainly distributed in organelles related to vesicle transport, and involved in the molecular function of binding protein. And Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DAPs were committed to regulating the synthesis of proteasome and spliceosome. Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that ARPC1B, PDAP1, and SEC61B can be used as special markers to distinguish GMSCs from PDLSCs. This research contributes to explaining the molecular mechanism and promoting the clinical application of tissue regeneration of GMSCs and PDLSCs.     

Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3‐dioxygenase1

Although human amniotic fluid does contain different populations of foetal‐derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in vitro expanded, amniotic fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second‐trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon (IFN)‐γ, including induction of the immunomodulatory enzyme indoleamine 2,3‐dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN‐γ–treated fHASCs caused significantly decreased T‐cell proliferation and increased frequency in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells. Both effects required an intact IDO1 function and were cell contact‐independent. An unprecedented finding in our study was that purified vesicles from IFN‐γ–treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC‐like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4⁺ CD25⁺ Foxp3⁺ T cells in graft‐draining lymph nodes from recipient mice. Thus fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo.     

Therapeutic potential of periodontal ligament stem cells

Inflammatory periodontal disease known as periodontitis is one of the most common conditions that affect human teeth and often leads to tooth loss. Due to the complexity of the periodontium, which is composed of several tissues, its regeneration and subsequent return to a homeostatic state is challenging with the therapies currently available. Cellular therapy is increasingly becoming an alternative in regenerative medicine/dentistry, especially therapies using mesenchymal stem cells, as they can be isolated from a myriad of tissues. Periodontal ligament stem cells (PDLSCs) are probably the most adequate to be used as a cell source with the aim of regenerating the periodontium. Biological insights have also highlighted PDLSCs as promising immunomodulator agents. In this review, we explore the state of knowledge regarding the properties of PDLSCs, as well as their therapeutic potential, describing current and future clinical applications based on tissue engineering techniques.     

Enamel matrix derivative enhances the proliferation and osteogenic differentiation of human periodontal ligament stem cells on the titanium implant surface

Periodontal ligament stem cells (PDLSCs) have mesenchymal-stem-cells-like qualities, and are considered as one of the candidates of future clinical application in periodontal regeneration therapy. Enamel matrix derivative (EMD) is widely used in promoting periodontal regeneration. However, the effects of EMD on the proliferation and osteogenic differentiation of human PDLSCs grown on the Ti implant surface are still no clear. Therefore, this study examined the effects of EMD on human PDLSCs in vitro. Human PDLSCs were isolated from healthy participants, and seeded on the surface of Ti implant disks and stimulated with various concentrations of EMD. Cell proliferation was determined with Cell Counting Kit-8 (CCK-8). The osteogenic differentiation of PDLSCs was evaluated by the measurement of alkaline phosphatase (ALP) activity, Alizarin red staining, and real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The results indicated that EMD at concentrations (5–60 µg/ml) increased the viability and proliferation of PDLSCs. The treatment with 30 and 60 µg/ml of EMD significantly elevated ALP activity, augmented mineralized nodule formation and calcium deposition, and upregulated the mRNA and protein levels of Runx-2 and osteocalcin (OCN) in the PDLSCs grown on the Ti surface. Further investigation found that EMD treatment did not change the protein levels of phosphatidylinositol-3-kinase (PI3K), p-PI3K, Akt and mTOR, but significantly upregulated the phosphorylated levels of Akt and mTOR. Collectively, these results suggest that EMD stimulation can promote the proliferation and osteogenic differentiation of PDLSCs grown on Ti surface, which is possibly associated with the activation of Akt/mTOR signaling pathway.     

Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses

Pharmacological inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine gestation results in fetal allograft rejection and blocks the ability of murine CD8(+) dendritic cells to suppress delayed-type hypersensitivity responses to tumor-associated peptide Ags. These observations suggest that cells expressing IDO inhibit T cell responses in vivo. To directly evaluate the hypothesis that cells expressing IDO inhibit T cell responses, we prepared IDO-transfected cell lines and transgenic mice overexpressing IDO and assessed allogeneic T cell responses in vitro and in vivo. T cells cocultured with IDO-transfected cells did not proliferate but expressed activation markers. The potency of allogeneic T cell responses was reduced significantly when mice were preimmunized with IDO-transfected cells. In addition, adoptive transfer of alloreactive donor T cells yielded reduced numbers of donor T cells when injected into IDO-transgenic recipient mice. These outcomes suggest that genetically enhanced IDO activity inhibited T cell proliferation in vitro and in vivo. Genetic manipulation of IDO activity may be of therapeutic utility in suppressing undesirable T cell responses.