Loss of Grem1-lineage chondrogenic progenitor cells causes osteoarthritis.

Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 (Grem1) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1-lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1-lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1-lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA.

Loss of Grem1-articular cartilage progenitor cells causes osteoarthritis.

Osteoarthritis (OA), which carries an enormous disease burden across the world, is characterised by irreversible degeneration of articular cartilage (AC), and subsequently bone. The cellular cause of OA is unknown. Here, using lineage tracing in mice, we show that the BMP-antagonist Gremlin 1 (Grem1) marks a novel chondrogenic progenitor (CP) cell population in the articular surface that generates joint cartilage and subchondral bone during development and adulthood. Notably, this CP population is depleted in injury-induced OA, and with age. OA is also induced by toxin-mediated ablation of Grem1 CP cells in young mice. Transcriptomic analysis and functional modelling in mice revealed articular surface Grem1-lineage cells are dependent on Foxo1; ablation of Foxo1 in Grem1-lineage cells led to early OA. This analysis identified FGFR3 signalling as a therapeutic target, and injection of its activator, FGF18, caused proliferation of Grem1-lineage CP cells, increased cartilage thickness, and reduced OA pathology. We propose that OA arises from the loss of CP cells at the articular surface secondary to an imbalance in progenitor cell homeostasis and present a new progenitor population as a locus for OA therapy.

Generation of Neural Crest-Like Cells From Human Periodontal Ligament Cell-Derived Induced Pluripotent Stem Cells.

Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue-derived periodontal ligament (PDL) iPSC to differentiate into neural crest-like cells (NCLC) relative to iPSC generated from a non-neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC- and FF iPSC-derived NCLC, which highly expressed HNK1. A high proportion of the HNK1-positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1-positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell-associated markers than the PDL and FF HNK1-negative populations. Interestingly, the HNK1-positive cells derived from the PDL-iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1-positive cells derived from the FF-iPSC. Our work suggests that HNK1-enriched NCLC from neural crest tissue-derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1-low population and non-neural crest iPSC-derived NCLC. J. Cell. Physiol. 232: 402-416, 2017. © 2016 Wiley Periodicals, Inc.

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

Differentiation of iPSC to Mesenchymal Stem-Like Cells and Their Characterization.

Mesenchymal stem cells (MSC) are a unique population of adult stem cells that have the capacity to differentiate into numerous cell types as well as the ability to modulate the immune system. As such, MSC represent a promising stem cell population for use in the clinical treatment of a range of disorders involving tissue regeneration as well as the immune system. The lack of accessibility to MSC is currently limiting the use of MSC in mainstream clinical treatment strategies. It is therefore imperative for the future success of stem cell-based treatment approaches that are more reliable, and accessible sources of MSC are identified. The present chapter describes a method for generating MSC-like cells from induced pluripotent stem cells (iPSC), with equivalent growth and functional properties to parental MSC populations.

Cementum and Periodontal Ligament Regeneration.

The unique anatomy and composition of the periodontium make periodontal tissue healing and regeneration a complex process. Periodontal regeneration aims to recapitulate the crucial stages of wound healing associated with periodontal development in order to restore lost tissues to their original form and function and for regeneration to occur, healing events must progress in an ordered and programmed sequence both temporally and spatially, replicating key developmental events. A number of procedures have been employed to promote true and predictable regeneration of the periodontium. Principally, the approaches are based on the use of graft materials to compensate for the bone loss incurred as a result of periodontal disease, use of barrier membranes for guided tissue regeneration and use of bioactive molecules. More recently, the concept of tissue engineering has been integrated into research and applications of regenerative dentistry, including periodontics, to aim to manage damaged and lost oral tissues, through reconstruction and regeneration of the periodontium and alleviate the shortcomings of more conventional therapeutic options. The essential components for generating effective cellular based therapeutic strategies include a population of multi-potential progenitor cells, presence of signalling molecules/inductive morphogenic signals and a conductive extracellular matrix scaffold or appropriate delivery system. Mesenchymal stem cells are considered suitable candidates for cell-based tissue engineering strategies owing to their extensive expansion rate and potential to differentiate into cells of multiple organs and systems. Mesenchymal stem cells derived from multiple tissue sources have been investigated in pre-clinical animal studies and clinical settings for the treatment and regeneration of the periodontium.

Ectopic Bone Formation by Mesenchymal Stem Cells Derived from Human Term Placenta and the Decidua.

Mesenchymal stem cells (MSCs) are one of the most attractive cell types for cell-based bone tissue repair applications. Fetal-derived MSCs and maternal-derived MSCs have been isolated from chorionic villi of human term placenta and the decidua basalis attached to the placenta following delivery, respectively. Chorionic-derived MSCs (CMSCs) and decidua-derived MSCs (DMSCs) generated in this study met the MSCs criteria set by International Society of Cellular Therapy. These criteria include: (i) adherence to plastic; (ii) >90% expression of CD73, CD105, CD90, CD146, CD44 and CD166 combined with <5% expression of CD45, CD19 and HLA-DR; and (iii) ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. In vivo subcutaneous implantation into SCID mice showed that both bromo-deoxyuridine (BrdU)-labelled CMSCs and DMSCs when implanted together with hydroxyapatite/tricalcium phosphate particles were capable of forming ectopic bone at 8-weeks post-transplantation. Histological assessment showed expression of bone markers, osteopontin (OPN), osteocalcin (OCN), biglycan (BGN), bone sialoprotein (BSP), and also a marker of vasculature, alpha-smooth muscle actin (α-SMA). This study provides evidence to support CMSCs and DMSCs as cellular candidates with potent bone forming capacity.

Novel basic helix-loop-helix transcription factor hes4 antagonizes the function of twist-1 to regulate lineage commitment of bone marrow stromal/stem cells.

Basic helix-loop-helix (bHLH) transcription factors are pivotal regulators of cellular differentiation and development. The bHLH factor, Twist-1 has previously been found to control bone marrow stromal/stem cells (BMSC) self-renewal, life span, and differentiation, however not much is known about its mechanism of action. In this study, we have discovered a novel Twist-1 regulated bHLH gene, Hes4, expressed in humans, but not in mice. Its closest homologue in both humans and mice is Hes1. Overexpression and knockdown studies demonstrated that Hes4 promotes osteogenesis resulting in an increase in Runx2, osteocalcin, osteopontin, and bone sialoprotein expression. Conversely, Hes4 was found to inhibit adipogenesis accompanied by a decrease in PPARγ2, adiponectin, and adipsin expression. In vitro studies indicate that Hes4 employs a mechanism to counteract the negative function of Twist-1 on osteogenesis by binding to Twist-1 and inhibiting the ability of Twist-1 to bind and inhibit Runx2. In vivo chromatin immunoprecipitation and in vitro reporter assays illustrated that Runx2 recruitment to the osterix promoter, was found to be enhanced in the presence of Hes4 and inhibited in the presence of Twist-1. Therefore, Hes4 antagonizes the function of Twist-1 to regulate lineage commitment of BMSC. These studies highlight the potential differences in molecular mechanisms that regulate BMSC osteogenic differentiation between human and mouse.

Semaphorin 3A induces mesenchymal-stem-like properties in human periodontal ligament cells.

Periodontal ligament stem cells (PDLSCs) have recently been proposed as a novel option in periodontal regenerative therapy. However, one of the issues is the difficulty of stably generating PDLSCs because of the variation of stem cell potential between donors. Here, we show that Semaphorin 3A (Sema3A) can induce mesenchymal-stem-like properties in human periodontal ligament (PDL) cells. Sema3A expression was specifically observed in the dental follicle during tooth development and in parts of mature PDL tissue in rodent tooth and periodontal tissue. Sema3A expression levels were found to be higher in multipotential human PDL cell clones compared with low-differentiation potential clones. Sema3A-overexpressing PDL cells exhibited an enhanced capacity to differentiate into both functional osteoblasts and adipocytes. Moreover, PDL cells treated with Sema3A only at the initiation of culture stimulated osteogenesis, while Sema3A treatment throughout the culture had no effect on osteogenic differentiation. Finally, Sema3A-overexpressing PDL cells upregulated the expression of embryonic stem cell markers (NANOG, OCT4, and E-cadherin) and mesenchymal stem cell markers (CD73, CD90, CD105, CD146, and CD166), and Sema3A promoted cell division activity of PDL cells. These results suggest that Sema3A may possess the function to convert PDL cells into mesenchymal-stem-like cells.

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.

Fabrication of a novel triphasic and bioactive ceramic and evaluation of its in vitro and in vivo cytocompatibility and osteogenesis.

We report, for the first time, the synthesis of a novel triphasic and crystalline bioactive ceramic (MSM-10) with the ability to simultaneously release three types of bioactive ions (strontium (Sr), silicon (Si) and magnesium (Mg)) to the surrounding microenvironment. An MSM-10 powder with a nominal composition (wt%) of 54 Mg2SiO4, 36 Si3Sr5 and 10 MgO was prepared by the sol-gel method and fabricated as porous scaffolds using the foam replication method. The effects of the different amounts of the phases in the ceramics on the mechanical and physical properties of the scaffolds as well as their in vitro and in vivo behaviors were comprehensively investigated. Biphasic calcium phosphate (BCP, ß-tricalcium phosphate (60 wt%)/hydroxyapatite (40 wt%)) scaffolds were used as the control material. The attachment, morphology, proliferation and differentiation of primary human osteoblasts (HOBs) were investigated after cell culturing on the various scaffolds. In vitro cytotoxicity (ISO/EN 10993-5) results not only indicated the biocompatibility of MSM-10, but also its positive effects on inducing the proliferation of HOBs. Our results showed significant enhancement in osteogenic gene expression levels (Runx2, osteocalcin, osteopontin and bone sialoprotein), when HOBs were cultured on MSM-10, compared to those for BCP and other generated ceramic scaffolds. For the in vivo studies, the different types of the materials were seeded with cultured human mesenchymal stem cells (hMSC) and then subcutaneously transplanted into the dorsal surface of eight-week-old immunocompromised (NOD/SCID) mice. MSM-10 demonstrated a significant amount of new bone formation compared to the other groups tested with no macroscopic signs of inflammation or toxicity in the tissue surrounding the implants. The novel MSM-10 ceramic presents promising potential for bone regeneration in orthopaedic and maxillofacial applications.

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.

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 Sharpey's 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.

EphB4 enhances the process of endochondral ossification and inhibits remodeling during bone fracture repair.

Previous reports have identified a role for the tyrosine kinase receptor EphB4 and its ligand, ephrinB2, as potential mediators of both bone formation by osteoblasts and bone resorption by osteoclasts. In the present study, we examined the role of EphB4 during bone repair after traumatic injury. We performed femoral fractures with internal fixation in transgenic mice that overexpress EphB4 under the collagen type 1 promoter (Col1-EphB4) and investigated the bone repair process up to 12 weeks postfracture. The data indicated that Col1-EphB4 mice exhibited stiffer and stronger bones after fracture compared with wild-type mice. The fractured bones of Col1-EphB4 transgenic mice displayed significantly greater tissue and bone volume 2 weeks postfracture compared with that of wild-type mice. These findings correlated with increased chondrogenesis and mineral formation within the callus site at 2 weeks postfracture, as demonstrated by increased safranin O and von Kossa staining, respectively. Interestingly, Col1-EphB4 mice were found to possess significantly greater numbers of clonogenic mesenchymal stromal progenitor cells (CFU-F), with an increased capacity to form mineralized nodules in vitro under osteogenic conditions, when compared with those of the wild-type control mice. Furthermore, Col1-EphB4 mice had significantly lower numbers of TRAP-positive multinucleated osteoclasts within the callus site. Taken together, these observations suggest that EphB4 promotes endochondral ossification while inhibiting osteoclast development during callus formation and may represent a novel drug target for the repair of fractured bones.

Isolation and characterization of mesenchymal stem cell-like cells from healthy and inflamed gingival tissue: potential use for clinical therapy.

AIM: Postnatal mesenchymal stem cell (MSC)-like cells have previously been isolated and ex vivo-expanded from healthy gingival tissues. The aim of this research was to isolate and characterize MSC-like cells from inflamed gingival tissues and determine whether they retain the characteristics of MSC-like cells from healthy gingival tissues. MATERIALS & METHODS: Fifteen clonal lines of MSC-like cells from three healthy gingival tissues (GMSC-H) and fifteen from three inflamed gingival tissues (GMSC-I) were generated. Bulk-cultured cell lines from healthy and inflamed gingival tissues were also established. In vitro and in vivo characterization studies of GMSC-Is were performed relative to GMSC-Hs. RESULTS: The incidence of clonogenic colony forming units-fibroblast was comparable between healthy and inflamed gingival tissues. GMSC-H and GMSC-I clones expressed MSC-associated markers CD44, CD73, CD90, CD105 and CD166. While the population doubling capacity of GMSC-Is was reduced compared with GMSC-Hs, both populations displayed a similar capacity to undergo osteogenic, adipogenic and chondrogenic differentiation in vitro. Following subcutaneous implantation in NOD/SCID mice, both GMSC-Hs and GMSC-Is formed dense connective tissue-like structures in vivo resembling natural gingival tissue. CONCLUSION: MSC-like populations exist within inflamed gingival tissue that are functionally equivalent to MSC-like cells derived from healthy gingival tissue. Given the relative abundance of inflamed gingival tissue and ease of accessibility, MSC-like cells from inflamed gingival tissues represent a newly identified population of postnatal stem cells with immense potential in tissue engineering applications.

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.

Effect of coating Straumann Bone Ceramic with Emdogain on mesenchymal stromal cell hard tissue formation.

Periodontal tissue engineering requires a suitable biocompatible scaffold, cells with regenerative capacity, and instructional molecules. In this study, we investigated the capacity of Straumann Bone Ceramic coated with Straumann Emdogain, a clinical preparation of enamel matrix protein (EMP), to aid in hard tissue formation by post-natal mesenchymal stromal cells (MSCs) including bone marrow stromal cells (BMSCs) and periodontal ligament fibroblasts (PDLFs). MSCs were isolated and ex vivo-expanded from human bone marrow and periodontal ligament and, in culture, allowed to attach to Bone Ceramic in the presence or absence of Emdogain. Gene expression of bone-related proteins was investigated by real time RT-PCR for 72 h, and ectopic bone formation was assessed histologically in subcutaneous implants of Bone Ceramic containing MSCs with or without Emdogain in NOD/SCID mice. Alkaline phosphatase activity was also assessed in vitro, in the presence or absence of Emdogain. Collagen-I mRNA was up-regulated in both MSC populations over the 72-h time course with Emdogain. Expression of BMP-2 and the osteogenic transcription factor Cbfa-1 showed early stimulation in both MSC types after 24 h. In contrast, expression of BMP-4 was consistently down-regulated in both MSC types with Emdogain. Up-regulation of osteopontin and periostin mRNA was restricted to BMSCs, while higher levels of bone sialoprotein-II were observed in PDLFs with Emdogain. Furthermore, alkaline phosphatase activity levels were reduced in both BMSCs and PDLFs in the presence of Emdogain. Very little evidence was found for ectopic bone formation following subcutaneous implantation of MSCs with Emdogain-coated or -uncoated Bone Ceramic in NOD/SCID mice. The early up-regulation of several important bone-related genes suggests that Emdogain may have a significant stimulatory effect in the commitment of mesenchymal cells to osteogenic differentiation in vitro. While Emdogain inhibited AP activity and appeared not to induce ectopic bone formation, longer-term studies are required to determine whether it promotes the final stages of osteoblast formation and mineralization at gene and protein levels. While used in clinical applications, whether Emdogain and other commercial preparations of EMPs truly possess the capacity to induce the regeneration of bone or other components of the periodontium remains to be established.

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.

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.

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.