Danijela Menicanin

Immunomodulatory properties of human periodontal ligament stem cells.

Tissue engineering utilizing periodontal ligament stem cells (PDLSCs) has recently been proposed for the development of new periodontal regenerative therapies. Although the use of autologous PDLSC transplantation eliminates the potential of a significant host immune response against the donor cells, it is often difficult to generate enough PDLSCs from one donor source due to the variation of stem cell potential between donors and disease state of each patient. In this study, we examined the immunomodulatory properties of PDLSCs as candidates for new allogeneic stem cell‐based therapies. Human PDLSCs displayed cell surface marker characteristics and differentiation potential similar to bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). PDLSCs, BMSSCs, and DPSCs inhibited peripheral blood mononuclear cell (PBMNC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction (MLR). Interestingly, gingival fibroblasts (GFs) also suppressed allogeneic PBMNC proliferation under both assay conditions. PDLSCs, BMSSCs, DPSCs, and GFs exhibited non‐cell contact dependent suppression of PBMNC proliferation in co‐cultures using transwells. Furthermore, conditioned media (CM) derived from each cell type pretreated with IFN‐γ partially suppressed PBMNC proliferation when compared to CMs without IFN‐γ stimulation. In all of these mesenchymal cell types cultured with activated PBMNCs, the expression of TGF‐β1, hepatocyte growth factor (HGF) and indoleamine 2, 3‐dioxygenase (IDO) was upregulated while IDO expression was upregulated following stimulation with IFN‐γ. These results suggest that PDLSCs, BMSSCs, DPSCs, and GFs possess immunosuppressive properties mediated, in part, by soluble factors, produced by activated PBMNCs. J. Cell. Physiol. 219: 667–676, 2009.

Clinical utility of stem cells for periodontal regeneration

The aim of this review is to discuss the clinical utility of stem cells in periodontal regeneration by reviewing relevant literature that assesses the periodontal-regenerative potential of stem cells. We considered and described the main stem cell populations that have been utilized with regard to periodontal regeneration, including bone marrow-derived mesenchymal stem cells and the main dental-derived mesenchymal stem cell populations: periodontal ligament stem cells, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, stem cells from apical papilla and dental follicle precursor cells. Research into the use of stem cells for tissue regeneration has the potential to significantly influence periodontal treatment strategies in the future.

EZH2 and KDM6A act as an epigenetic switch to regulate mesenchymal stem cell lineage specification.

The methyltransferase, Enhancer of Zeste homology 2 (EZH2), trimethylates histone 3 lysine 27 (H3K27me3) on chromatin and this repressive mark is removed by lysine demethylase 6A (KDM6A). Loss of these epigenetic modifiers results in developmental defects. We demonstrate that Ezh2 and Kdm6a transcript levels change during differentiation of multipotential human bone marrow‐derived mesenchymal stem cells (MSC). Enforced expression of Ezh2 in MSC promoted adipogenic in vitro and inhibited osteogenic differentiation potential in vitro and in vivo, whereas Kdm6a inhibited adipogenesis in vitro and promoted osteogenic differentiation in vitro and in vivo. Inhibition of EZH2 activity and knockdown of Ezh2 gene expression in human MSC resulted in decreased adipogenesis and increased osteogenesis. Conversely, knockdown of Kdm6a gene expression in MSC leads to increased adipogenesis and decreased osteogenesis. Both Ezh2 and Kdm6a were shown to affect expression of master regulatory genes involved in adipogenesis and osteogenesis and H3K27me3 on the promoters of master regulatory genes. These findings demonstrate an important epigenetic switch centered on H3K27me3 which dictates MSC lineage determination. Stem Cells 2014;32:802–815

Generation of functional mesenchymal stem cells from different induced pluripotent stem cell lines.

The therapeutic potential of mesenchymal stem cells (MSC) has highlighted the need for identifying easily accessible and reliable sources of these cells. An alternative source for obtaining large populations of MSC is through the controlled differentiation of induced pluripotent stem cells (iPSC). In the present study, colonies of iPSC were cultured in MSC culture media for 2 weeks. Serial passaging then selected for fast growing MSC-like cells with a typical fibroblastic morphology and the capacity to proliferate on standard culture flasks without feeder cells. MSC-like cells were developed from iPSC lines arising from three different somatic tissues: gingiva, periodontal ligament (PDL), and lung. The iPSC-MSC like cells expressed key MSC-associated markers (CD73, CD90, CD105, CD146, and CD166) and lacked expression of pluripotent markers (TRA160, TRA181, and alkaline phosphatase) and hematopoietic markers (CD14, CD34, and CD45). In vitro iPSC-MSC-like cells displayed the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes. In vivo subcutaneous implantation of the iPSC-MSC-like cells into NOD/SCID mice demonstrated that only the PDL-derived iPSC-MSC-like cells exhibited the capacity to form mature mineralized structures which were histologically similar to mature bone. These findings demonstrate that controlled induction of iPSC into fibroblastic-like cells that phenotypically and functionally resemble adult MSC is an attractive approach to obtain a readily available source of progenitor cells for orthopedic and dental-related tissue-engineering applications. However, a detailed characterization of the iPSC-MSC-like cells will be important, as MSC-like cells derived from different iPSC lines exhibit variability in their differentiation capacity.

Genomic Profiling of Mesenchymal Stem Cells

Mesenchymal stem/stromal cells (MSC) are an accessible source of precursor cells that can be expanded in vitro and used for tissue regeneration for different clinical applications. The advent of microarray technology has enabled the monitoring of individual and global gene expression patterns across multiple cell populations. Thus, genomic profiling has fundamentally changed our capacity to characterize MSCs, identify potential biomarkers and determined key molecules regulating biological processes involved in stem cell survival, growth and development. Numerous studies have now examined the genomic profiles of MSCs derived from different tissues that exhibit varying levels of differentiation and proliferation potentials. The knowledge gained from these studies will help improve our understanding of the cellular signalling pathways involved in MSC growth, survival and differentiation, and may aid in the development of strategies to improve the tissue regeneration potential of MSCs for different clinical indications. The present review summarizes studies characterizing the gene expression profile of MSCs.

Mesenchymal Stem Cells from iPS Cells Facilitate Periodontal Regeneration

Mesenchymal stem cells (MSC) have been considered as a potential therapy for the treatment of periodontal defects arising from periodontitis. However, issues surrounding their accessibility and proliferation in culture significantly limit their ability to be used as a mainstream treatment approach. It is therefore important that alternative, easily accessible, and safe populations of stem cells be identified. Controlled induction of induced pluripotent stem cells (iPSC) into MSC-like cells is emerging as an attractive source for obtaining large populations of stem cells for regenerative medicine. We have successfully induced iPSC to differentiate into MSC-like cells. The MSC-like cells generated satisfied the International Society of Cellular Therapy’s minimal criteria for defining multipotent MSC, since they had plastic adherent properties, expressed key MSC-associated markers, and had the capacity to undergo tri-lineage differentiation. Importantly, the resulting iPSC-MSC-like cells also had the capacity, when implanted into periodontal defects, to significantly increase the amount of regeneration and newly formed mineralized tissue present. Our results demonstrate, for the first time, that MSC derived from iPSC have the capacity to aid periodontal regeneration and are a promising source of readily accessible stem cells for use in the clinical treatment of periodontitis.

Heat Shock Protein-90 beta Is Expressed at the Surface of Multipotential Mesenchymal Precursor Cells: Generation of a Novel Monoclonal Antibody, STRO-4, With Specificity for Mesenchymal Precursor Cells From Human and Ovine Tissues

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

Identification of a common gene expression signature associated with immature clonal mesenchymal cell populations derived from bone marrow and dental tissues.

Mesenchymal stem/stromal cell-like populations derived from adult bone marrow (BMSC), dental pulp (DPSC), and periodontal ligament (PDLSC) have the ability to differentiate into cells of mesenchymal and non-mesenchymal tissues in vitro and in vivo. However, culture-expanded MSC-like populations are a heterogeneous mix of stem/committed progenitor cells that exhibit altered growth and developmental potentials. In the present study we isolated and characterized clonal populations of BMSCs, DPSCs, and PDLSCs to identify potential biomarkers associated with long-lived multipotential stem cells. Microarray analysis was used to compare the global gene expression profiles of high growth/multipotential clones with low growth potential cell clones derived from 3 stromal tissues. Cross-comparison analyses of genes expressed by high growth/multipotential clones derived from bone marrow, dental pulp, and periodontal ligament identified 24 genes that are differentially up-regulated in all tissues. Notably, the transcription factors, E2F2, PTTG1, TWIST-1, and transcriptional cofactor, LDB2, each with critical roles in cell growth and survival, were highly expressed in all stem cell populations examined. These findings provide a model system for identifying a common molecular fingerprint associated with immature mesenchymal stem-like cells from different organs and implicate a potential role for these genes in MSC growth and development.

Periodontal-Ligament-Derived Stem Cells Exhibit the Capacity for Long-Term Survival, Self-Renewal, and Regeneration of Multiple Tissue Types in Vivo

Primary periodontal ligament stem cells (PDLSCs) are known to possess multidifferentiation potential and exhibit an immunophenotype similar to that described for bone-marrow-derived mesenchymal stem cells. In the present study, bromo-deoxyuridine (BrdU)-labeled ovine PDLSCs implanted into immunodeficient mice survived after 8 weeks post-transplantation and exhibited the capacity to form bone/cementum-like mineralized tissue, ligament structures similar to Sharpeys fibers with an associated vasculature. To evaluate self-renewal potential, PDLSCs were recovered from harvested primary transplants 8 weeks post-transplantation that exhibit an immunophenotype and multipotential capacity comparable to primary PDLSCs. The re-derived PDLSCs isolated from primary transplants were implanted into secondary ectopic xenogeneic transplants. Histomorphological analysis demonstrated that four out of six donor re-derived PDLSC populations displayed a capacity to survive and form fibrous ligament structures and mineralized tissues associated with vasculature in vivo, although at diminished levels in comparison to primary PDLSCs. Further, the capacity for long-term survival and the potential role of PDLSCs in dental tissue regeneration were determined using an ovine preclinical periodontal defect model. Autologous ex vivo-expanded PDLSCs that were prelabeled with BrdU were seeded onto Gelfoam(®) scaffolds and then transplanted into fenestration defects surgically created in the periodontium of the second premolars. Histological assessment at 8 weeks post-implantation revealed surviving BrdU-positive PDLSCs associated with regenerated periodontium-related tissues, including cementum and bone-like structures. This is the first report to demonstrate the self-renewal capacity of PDLSCs using serial xenogeneic transplants and provides evidence of the long-term survival and tissue contribution of autologous PDLSCs in a preclinical periodontal defect model.

Stem cells, tissue engineering and periodontal regeneration

The aim of this review is to discuss the clinical utility of stem cells in periodontal regeneration by reviewing relevant literature that assesses the periodontal-regenerative potential of stem cells. We consider and describe the main stem cell populations that have been utilized with regard to periodontal regeneration, including bone marrow-derived mesenchymal stem cells and the main dental-derived mesenchymal stem cell populations: periodontal ligament stem cells, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, stem cells from apical papilla and dental follicle precursor cells. Research into the use of stem cells for tissue regeneration has the potential to significantly influence periodontal treatment strategies in the future.