Neuropilin Controls Endothelial Differentiation by Mesenchymal Stem Cells From the Periodontal Ligament

Background: Periodontal ligament (PDL) has been reported to be a source of mesenchymal stem cells (MSCs).New vascular networks from undifferentiated cells are essential for repair/regeneration of specialized tissues, including PDL. The current study aims to determine potential of CD105⁺‐enriched cell subsets of periodontal ligament cells (PDLSCs) to differentiate into endothelial cell (EC)‐like cells and to give insights into the mechanism involved. Methods: CD105⁺‐enriched PDLSCs were induced to EC differentiation by endothelial growth medium 2 (EGM‐2) for 3, 7, 14, and 21 days, with mRNA/protein levels and functional activity assessed by: 1) real‐time polymerase chain reaction; 2) Western blotting; 3) fluorescence‐activated cell sorting; 4) immunohistochemistry; 5) immunofluorescence; 6) matrigel; and 7) small interfering RNA assays. Results: Data analyses demonstrated that EGM‐2 treated PDLSCs presented increased expression of EC markers, including: 1) CD105; 2) kinase domain‐containing receptor; and 3) Ulex europaeus agglutinin 1, and were able to form cord/tube‐like structures. Gene and protein expression analysis showed that neuropilin 2 (NRP2), a key factor for vascular development, was significantly downregulated during EC differentiation. NRP2 was constitutively expressed in mouse PDL tissues by immunohistochemistry analysis, and NRP2 knockdown in CD105⁺‐enriched PDLSCs resulted in increased cord/tube‐like structures in a matrigel assay. Conclusion: These findings demonstrated the potential of CD105⁺‐enriched PDLSCs to support angiogenesis, and NRP2 as a pivotal factor regulating this process.     

Stem cell properties of human periodontal ligament cells

BACKGROUND AND OBJECTIVE: Stem cells have been used for regenerative therapies in various fields. The proportion of cells that possess stem cell properties in human periodontal ligament (PDL) cells is not yet well understood. In this study, we quantitatively characterized human PDL cells to clarify their stem cell properties, including self-renewal, multipotency, and stem cell marker expression. MATERIAL AND METHODS: PDL cells were obtained from extracted premolar or wisdom teeth, following which a proliferation assay for self-renewal, a differentiation assay for multipotency, immunostaining for STRO-1, and fluorescence-activated cell sorter (FACS) analysis for stem cell markers (including CD105, CD166, and STRO-1) were performed. RESULTS: Approximately 30% of 400 PDL cells were found to possess replicative potential and formed single-cell colonies, and 30% of these colonies displayed positive staining for STRO-1, 20% differentiated into adipocytes and 30% differentiated into osteoblasts. FACS analysis revealed that PDL cells, including cell populations, expressed the stem cell markers CD105, CD166, and STRO-1. CONCLUSION: The findings of this study indicated that PDL cells possess crucial stem cell properties, such as self-renewal and multipotency, and express the mesenchymal stem cell markers CD105, CD166, and STRO-1 on their cell surface, although there were some variations. Thus, PDL cells can be used for periodontal regenerative procedures.     

Cell surface markers CD44 and CD166 localized specific populations of salivary acinar cells

Oral Diseases (2012) 18 , 162–168 Objective: Experimental approaches tested to date for functional restoration of salivary glands (SGs) are tissue engineering, gene transfer, and cell therapy. To further develop these therapies, identifying specific cell surface markers for the isolation of salivary acinar cells is needed. To test a panel of cell surface markers [used in the isolation of mesenchymal stem cells, (MSCs)] for the localization of salivary acinar cells. Materials: Human submandibular and parotid glands were immunostained with a panel of MSC markers and co‐localized with salivary acinar cell differentiation markers [α‐amylase, Na‐K‐2Cl cotransporter‐1, aquaporin‐5 (AQP5)]. Additional cell markers were also used, such as α‐smooth muscle actin (to identify myoepithelial cells), cytokeratin‐5 (basal ductal cells), and c‐Kit (progenitor cells). Results: CD44 identified serous acini, while CD166 identified mucous acini. Cytokeratin‐5 identified basal duct cells and 50% of myoepithelial cells. None of the remaining cell surface markers (Stro‐1, CD90, CD106, CD105, CD146, CD19, CD45, and c‐Kit) were expressed in any human salivary cell. Conclusions: CD44 and CD166 localized human salivary serous and mucous acinar cells, respectively. These two cell surface markers will be useful in the isolation of specific populations of salivary acinar cells.     

Periodontal Ligament Stem Cells Possess the Characteristics of Pericytes

Background: Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells. Methods: PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte‐like functions of PDLSCs were examined in co‐culture of PDLSCs and umbilical vein endothelial cells on a gel matrix. Results: Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146‐ and NG2‐positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary‐like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary‐like structure. Conclusions: PDLSCs possess pericyte‐like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell–based periodontal therapy.     

Effect of GDF-5 and BMP-2 on the expression of tendo/ligamentogenesis-related markers in human PDL-derived cells

Oral Diseases (2012) 18 , 206–212 Objectives: The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament‐derived cells was investigated with special reference to tendo/ligamentogenesis‐related markers. Materials and Methods: Effects of each factor were analyzed by quantitative PCR for scleraxis and tenomodulin and by western blotting for scleraxis. After exposure to those factors, STRO‐1‐positive and STRO‐1‐negative fractions of human periodontal ligament tissues were isolated with an immunomagnetic cell sorting system, and the expression of scleraxis in each fraction was analyzed by western blotting. Non‐separated crude cells were used as a control. Results: Growth differentiation factor 5 and bone morphogenetic protein 2 did not increase alkaline phosphatase activity in crude periodontal ligament‐derived cells. Growth differentiation factor 5, but not bone morphogenetic protein 2, increased the expression of scleraxis in crude, STRO‐1‐positive and STRO‐1‐negative periodontal ligament‐derived cells. The expression of scleraxis in STRO‐1‐positive periodontal ligament‐derived cells was significantly less compared to that in crude P2 and STRO‐1‐negative periodontal ligament‐derived cells. Conclusion: Growth differentiation factor 5 induced the expression of scleraxis and may enhance tendo/ligamentogenesis in human periodontal ligament‐derived cells. The expression of scleraxis was higher in STRO‐1‐negative fraction, suggesting more differentiated state of the cells.     

Establishing a technique for isolation and characterization of human periodontal ligament derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are extensively used in tissue regenerative procedures. One source of MSCs is the periodontal ligament (PDL) of teeth. Isolation of MSCs from extracted teeth is reasonably simple, being less invasive and presenting fewer ethical concerns than does the harvesting of MSC’s from other sites. The objectives of this study were to isolate and characterize the PDL stem cells (PDLSC) from healthy adults’ extracted teeth and then to characterize them by comparing them with bone-marrow derived MSCs (BMMSC).MethodsThe PDL tissue was scraped from the roots of freshly extracted teeth to enzymatically digest using collagenase. The cells were sub-cultured. Flow-cytometric analysis for the MSC surface-markers CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR was performed. To confirm the phenotype, total RNA was extracted to synthesize cDNA and which was then subjected to RT-PCR. The gene-expression for Oct4A, Sox2, NANOG and GAPDH was determined by gel-electrophoresis. To assess their multilineage potential, cells were cultured with osteogenic, chondrogenic and adipogenic medium and then stained by Alizarin-red, Alcian-blue and Oil-Red-O respectively. MSCs from the bone-marrow were processed similarly to serve as controls.ResultsThe cells isolated from extracted teeth expanded successfully. On flow-cytometric analysis, the cells were positive for CD73, CD90, CD105, CD166 and negative for CD34, CD45 and HLA-DR. The PDLSCs expressed Oct4A, Sox2, and NANOG mRNA with GAPDH expression. Cells cultured in the osteogenic, chondrogenic and adipogenic media stained positive for Alizarin-red, Alcian-blue and Oil- Red-O respectively. The surface marker expression and the trilineage differentiation characteristics were comparable to those of the BMMSCs.ConclusionsThe periodontal ligament tissue of extracted teeth is a potential source of therapeutically useful MSCs. Harvesting them is not invasive and are a promising source of MSC as the PDLSCs showed characteristics similar to those of the highly regarded MSC’s derived from bone-marrow.     

Stromal cell-derived factor-1 significantly induces proliferation, migration, and collagen type I expression in a human periodontal ligament stem cell subpopulation

Background: The pivotal role of chemokine stromal cell–derived factor‐1 (SDF‐1) in bone marrow mesenchymal stem cells recruitment and tissue regeneration has already been reported. However, its roles in human periodontal ligament stem cells (PDLSCs) remain unknown. PDLSCs are regarded as candidates for periodontal tissue regeneration and are used in stem cell–based periodontal tissue engineering. The expression of chemokine receptors on PDLSCs and the migration of these cells induced by chemokines and their subsequent function in tissue repair may be a crucial procedure for periodontal tissue regeneration. Methods: PDL tissues were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate single‐cell colonies by the limited‐dilution method. Immunocytochemical staining was used to detect the expression of the mesenchymal stem cell marker STRO‐1. Differentiation potentials were assessed by alizarin‐red staining and oil‐red O staining. The expression of SDF‐1 receptor CXCR4 was evaluated by real‐time polymerase chain reaction (PCR) and immunocytochemical staining. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay were used to determine the viability and proliferation of the PDLSC subpopulation. Expression of collagen type I and alkaline phosphatase was detected by real‐time PCR to determine the effect of SDF‐1 on cells differentiation. Results: Twenty percent of PDL single‐cell colonies expressed STRO‐1 positively, and this specific subpopulation was positive for CXCR4 and formed minerals and lipid vacuoles after 4 weeks induction. SDF‐1 significantly increased proliferation and stimulated the migration of this PDLSC subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing antibody could block cell proliferation and migration, suggesting that SDF‐1 exerted its effects on cells through CXCR4. SDF‐1 promoted collagen type I level significantly but had little effect on alkaline phosphatase level. Conclusion: SDF‐1 may have the potential of promoting periodontal tissue regeneration by the mechanism of guiding PDLSCs to destructive periodontal tissue, promoting their activation and proliferation and influencing the differentiation of these stem cells.     

干细胞表面标志物在牙髓干细胞中的表达

目的:探讨磁珠分选后培养的人牙髓干细胞的表面标记抗原随培养代数增加的变化情况。方法:改良组织块法培养的人牙髓细胞,至第2代（P2）时,用磁珠方法分选出STRO-1阳性细胞,用流式细胞术分别检测P2、P3、P4、P5、P6、P7、P8代的干细胞表面标志物CD73、CD90、CD105、 CD166、STRO-1。取P3代细胞,分别进行成骨诱导和成脂诱导。21 d后,分别行茜素红染色和油红O染色,观察矿化物形成情况和脂滴形成情况,同时以未诱导细胞为对照。结果:STRO-1在牙髓干细胞表面随代数增加而下降,CD73、CD90、CD105、 CD166的表达比较稳定,茜素红染色可见矿化结节形成,油红O染色显示形成大量脂滴。结论:STRO-1在牙髓干细胞表面随代数增加而下降,其他干细胞标志物比较稳定。     

FGF-2 induces proliferation of human periodontal ligament cells and maintains differentiation potentials of STRO-1(+)/CD146(+) periodontal ligament cells

The presence of human STRO-1⁺/CD146⁺ periodontal ligament (PDL) cells has been reported, but obtaining a large amount of these cells is difficult. The purpose of this study was to evaluate the percentages of STRO-1⁺/CD146⁺ cells in PDL cells and determine the effects of FGF-2 on the proliferation and multilineage differentiation potency of these cells. Human PDL (HPDL) cells were individually prepared from 15 extracted teeth. HPDL cells were cultured with or without FGF-2, and the percentages of STRO-1⁺/CD146⁺ cells in each HPDL cell culture was examined using FACSAria?. The STRO-1⁺/CD146⁺ cells were sorted with FACSAria?, and the mRNA expression and differentiation potency of the sorted cells were subsequently examined. The numbers of the STRO-1⁺/CD146⁺ cells in the FGF-2 cultures were significantly higher than those cultured in the absence of FGF-2. The sorted STRO-1⁺/CD146⁺ cells expressed mRNA of PDL markers and differentiated into adipocytes and osteoblast-like cells. The present study shows that FGF-2 augmented the proliferation of the STRO-1⁺/CD146⁺ cells in the HPDL cultures whilst retaining adipogenic and osteogenic differentiation potentials. Thus, it may be useful to culture HPDL cells with FGF-2 for the application of the human STRO-1⁺/CD146⁺ PDL cells in periodontal tissue regeneration.     

Alveolar Bone Resorption Induced by CD4+CD45RB High‐Density T‐Cell Transfer in Immunocompromised Mice

Background: The aims of this study are to determine whether the antigen‐inexperienced (naive, CD45RB high‐density) T‐cell (CD4⁺CD45RB^High T‐cell) transfer model is associated with alveolar bone resorption, to elucidate the local osteogenic/adipogenic potential of alveolar bone marrow stromal cells (ABCs) from T‐cell–transferred animals, and to investigate the systemic osteogenic potential by transplanting human periodontal ligament stem cells (hPDLSCs) into these animals. Methods: CD4⁺CD45RB^High and CD4⁺CD45RB^Low (antigen‐experienced [memory, CD45RB low‐density]) T cells were sorted and transferred into severe combined immunodeficiency (SCID) mice to induce inflammatory bowel disease–like syndrome (n = 8). hPDLSCs were transplanted into T‐cell–transferred SCID mice to examine ectopic cementum formation 8 weeks after T‐cell transfer. The mandibles and tibias of these mice were retrieved for microcomputed tomography (micro‐CT), histomorphometric analysis, and isolation of ABCs 16 weeks after T‐cell transfer. The in vitro osteogenic and adipogenic potentials of the ABCs were evaluated. Results: Histologic and micro‐CT analysis revealed that the transfer of CD4⁺CD45RB^High T‐cell subset was sufficient for alveolar bone resorption and affected the osteogenic/adipogenic potential of ABCs. Furthermore, it was found that CD4⁺CD45RB^High T‐cell–transferred animals have decreased systemic osteogenic potential, as evidenced using the in vivo ectopic hPDLSC transplantation model. Conclusion: CD4⁺CD45RB^High T‐cell transfer induced both alveolar bone resorption and reduced systemic osteogenic potential, with a concomitant downregulation of the osteogenic potential of ABCs.     

Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts

Wada N, Wang B, Lin N‐H, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival and periodontal ligament fibroblasts. J Periodont Res 2011; 46: 438–447. © 2011 John Wiley & Sons A/S Background and Objective: Human induced pluripotent stem (iPS) cells, which have similar properties to human embryonic stem (hES) cells, have been generated from neonatal and adult human dermal fibroblasts by reprogramming. iPS cells have high pluripotency and differentiation potential, and may be a potential autologous stem cell source for future regenerative therapy. Material and Methods: iPS cell lines from human gingival fibroblasts and, for the first time, from periodontal ligament fibroblasts, were generated by reprogramming using a retroviral transduction cocktail of OCT3/4, SOX2, KLF4 and c‐MYC. iPS induction was investigated through expression of the embryonic stem cell markers SSEA4, OCT4, NANOG, GCTM‐2, TG30 and TRA‐1‐60. Following in vitro differentiation, the expression of genes for differentiation markers for ectoderm (SOX1, PAX6), mesoderm [RUNX1, T(Brachyury)] and endoderm (GATA4, AFP) was assessed by real‐time RT‐PCR. The ability to form teratomas following implantation into mouse testes was assessed by histology. Results: Human gingival fibroblast‐ and periodontal ligament fibroblast‐derived iPS cells showed similar characteristics to hES cells. Both sets of iPS cells displayed colony morphology comparable to that of hES cells and expressed the hES cell‐associated cell‐surface antigens, SSEA3, SSEA4, GCTM‐2, TG30 (CD9) and Tra‐1‐60, and the hES cell marker genes, OCT4, NANOG and GDF3. These iPS cells showed differentiation potential to form embryoid bodies in vitro and expressed genes for endoderm, ectoderm and mesoderm. Teratoma formation following implantation into mouse testes was observed. Conclusion: These results demonstrate that iPS cells can be successfully generated from adult human gingival and periodontal ligament fibroblasts.