Thimios A. Mitsiadis

A biomarker-based mathematical model to predict bone-forming potency of human synovial and periosteal mesenchymal stem cells.

OBJECTIVE To develop a biomarker-based model to predict osteogenic potency of human mesenchymal stem cells (MSCs) from synovial membrane and periosteum. METHODS MSC populations were derived from adult synovium and periosteum. Phenotype analysis was performed by fluorescence-activated cell sorting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Telomere lengths were determined by Southern blot analysis. In vitro osteogenesis was assessed quantitatively by measurements of alkaline phosphatase activity and calcium deposits. To investigate bone formation in vivo, MSCs were seeded onto osteoinductive scaffolds and implanted subcutaneously in nude mice. Bone was assessed by histology, and the human origin investigated by in situ hybridization for human Alu genomic repeats. Quantitation was achieved by histomorphometry and real-time RT-PCR for human osteocalcin. Analysis at the single-cell level was performed with clonal populations obtained by limiting dilution. Multiple regressions were used to explore the incremental predictive value of the markers. RESULTS Periosteal MSCs had significantly greater osteogenic potency than did synovial MSCs inherent to the single cell. Bone was largely of human origin in vivo. Within the same tissue type, there was variability between different donors. To identify predictors of osteogenic potency, we measured the expression levels of osteoblast lineage genes in synovial and periosteal clonal MSCs prior to osteogenic treatment. We identified biomarkers that correlated with osteogenic outcome and developed a mathematical model based on type I collagen and osteoprotegerin expression that predicts the bone-forming potency of MSC preparations, independent of donor-related variables and tissue source. CONCLUSION Our findings indicate that our quality-control mathematical model estimates the bone-forming potency of MSC preparations for bone repair.

Cell Fate Determination During Tooth Development and Regeneration

Teeth arise from sequential and reciprocal interactions between the oral epithelium and the underlying cranial neural crest-derived mesenchyme. Their formation involves a precisely orchestrated series of molecular and morphogenetic events, and gives us the opportunity to discover and understand the nature of the signals that direct cell fates and patterning. For that reason, it is important to elucidate how signaling factors work together in a defined number of cells to generate the diverse and precise patterned structures of the mature functional teeth. Over the last decade, substantial research efforts have been directed toward elucidating the molecular mechanisms that control cell fate decisions during tooth development. These efforts have contributed toward the increased knowledge on dental stem cells, and observation of the molecular similarities that exist between tooth development and regeneration.

Bone morphogenetic protein-7 release from endogenous neural precursor cells suppresses the tumourigenicity of stem-like glioblastoma cells

Glioblastoma cells with stem-like properties control brain tumour growth and recurrence. Here, we show that endogenous neural precursor cells perform an anti-tumour response by specifically targeting stem-like brain tumour cells. In vitro, neural precursor cells predominantly express bone morphogenetic protein-7; bone morphogenetic protein-7 is constitutively released from neurospheres and induces canonical bone morphogenetic protein signalling in stem-like glioblastoma cells. Exposure of human and murine stem-like brain tumour cells to neurosphere-derived bone morphogenetic protein-7 induces tumour stem cell differentiation, attenuates stem-like marker expression and reduces self-renewal and the ability for tumour initiation. Neurosphere-derived or recombinant bone morphogenetic protein-7 reduces glioblastoma expansion from stem-like cells by down-regulating the transcription factor Olig2. In vivo, large numbers of bone morphogenetic protein-7-expressing neural precursors encircle brain tumours in young mice, induce canonical bone morphogenetic protein signalling in stem-like glioblastoma cells and can thereby attenuate tumour formation. This anti-tumour response is strongly reduced in older mice. Our results indicate that endogenous neural precursor cells protect the young brain from glioblastoma by releasing bone morphogenetic protein-7, which acts as a paracrine tumour suppressor that represses proliferation, self-renewal and tumour-initiation of stem-like glioblastoma cells.

Distinct mesenchymal progenitor cell subsets in the adult human synovium

OBJECTIVE To analyse the heterogeneity at the single-cell level of human mesenchymal progenitor cells from SM. METHODS Cell populations were enzymatically released from the knee joint synovium of adult human individuals. Single cell-derived clonal populations were obtained by limiting dilution and serially passaged to determine growth rates. Phenotypic analysis was carried out by flow cytometry. Replicative senescence was assessed by the senescence-associated beta-galactosidase staining. Telomere lengths were determined semiquantitatively by Southern blotting. Telomerase activity was measured using a real-time quantitative telomerase repeat amplification procedure. Culture-expanded clonal populations were subjected to in vitro differentiation assays to investigate their mesenchymal multipotency. RESULTS The 50 clones analysed displayed wide variations in the proliferation rates, even within the same donor sample. The time taken to reach 20 population doublings ranged from 44 to 130 days. The phenotype of the clones tested was compatible with that of mesenchymal stem cells. Mean telomere lengths ranged from 5.2 to 10.9 kb with positive linear trend with telomerase activity, but no correlation with proliferative rates or cell senescence. All clones tested were capable of chondrogenic and osteogenic differentiation, though with large variability in potency. In contrast, only 30% of the clones were adipogenic. CONCLUSIONS We report for the first time the co-existence, within the synovium, of progenitor cell subsets with distinct mesenchymal differentiation potency. Our findings further emphasize the need for strategies to purify cell populations with the clinically desired tissue formation potentials.

Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials

Numerous stem cell niches are present in the different tissues and organs of the adult human body. Among these tissues, dental pulp, entrapped within the ‘sealed niche’ of the pulp chamber, is an extremely rich site for collecting stem cells. In this study, we demonstrate that the isolation of human dental pulp stem cells by the explants culture method (hD‐DPSCs) allows the recovery of a population of dental mesenchymal stem cells that exhibit an elevated proliferation potential. Moreover, we highlight that hD‐DPSCs are not only capable of differentiating into osteoblasts and chondrocytes but are also able to switch their genetic programme when co‐cultured with murine myoblasts. High levels of MyoD expression were detected, indicating that muscle‐specific genes in dental pulp cells can be turned on through myogenic fusion, confirming thus their multipotency. A perivascular niche may be the potential source of hD‐DPSCs, as suggested by the consistent Ca2+ release from these cells in response to endothelin‐1 (ET‐1) treatment, which is also able to significantly increase cell proliferation. Moreover, response to ET‐1 has been found to be superior in hD‐DPSCs than in DPSCs, probably due to the isolation method that promotes release of stem/progenitor cells from perivascular structures. The ability to isolate, expand and direct the differentiation of hD‐DPSCs into several lineages, mainly towards myogenesis, offers an opportunity for the study of events associated with cell commitment and differentiation. Therefore, hD‐DPSCs display enhanced differentiation abilities when compared to DPSCs, and this might be of relevance for their use in therapy.

Dental Pulp Stem Cells, Niches, and Notch Signaling in Tooth Injury

Stem cells guarantee tissue repair and regeneration throughout life. The decision between cell self-renewal and differentiation is influenced by a specialized microenvironment called the ‘stem cell niche’. In the tooth, stem cell niches are formed at specific anatomic locations of the dental pulp. The microenvironment of these niches regulates how dental pulp stem cell populations participate in tissue maintenance, repair, and regeneration. Signaling molecules such as Notch proteins are important regulators of stem cell function, with various capacities to induce proliferation or differentiation. Dental injuries often lead to odontoblast apoptosis, which triggers activation of dental pulp stem cells followed by their proliferation, migration, and differentiation into odontoblast-like cells, which elaborate a reparative dentin. Better knowledge of the regulation of dental pulp stem cells within their niches in pathological conditions will aid in the development of novel treatments for dental tissue repair and regeneration.

Dental lamina as source of odontogenic stem cells: evolutionary origins and developmental control of tooth generation in gnathostomes.

This study considers stem cells for odontogenic capability in biological tooth renewal in the broad context of gnathostome dentitions and the derivation of them from oral epithelium. The location of the developmental site and cell dynamics of the dental lamina are parameters of a possible source for odontogenic epithelial stem cells, but the phylogenetic history is not known. Understanding the phylogenetic basis for stem cell origins throughout continuous tooth renewal in basal jawed vertebrates is the ultimate objective of this study. The key to understanding the origin and location of stem cells in the development of the dentition is sequestration of stem cells locally for programmed tooth renewal. We suggest not only the initial pattern differences in each dentate field but local control subsequently for tooth renewal within each family. The role of the specialized odontogenic epithelium (odontogenic band) is considered as that in which the stem cells reside and become partitioned. These regulate time, position and shape in sequential tooth production. New histological data for chondrichthyan fish show first a thickening of the oral epithelium (odontogenic band). After this, all primary and successive teeth are only generated deep to the oral epithelium from a dental lamina. In contrast, in osteichthyan fish the first teeth develop directly within the odontogenic band. In addition, successors are initiated at each tooth site in the predecessor tooth germ (without a dental lamina). We suggest that stem cells specified for each tooth family are set up and located in intermediate cells between the outer and inner dental epithelia.

Deletion of BMP7 Affects the Development of Bones, Teeth, and Other Ectodermal Appendages of the Orofacial Complex

Sequential and reciprocal epithelial-mesenchymal interactions govern the development of most tissues and organs of the craniofacial region. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of secreted signaling molecules that have long been implied to have a significant contribution in this process. However, evidence for such a role during craniofacial development is largely missing. Using a lacZ reporter mouse we mapped the spatiotemporal expression of BMP7 in the developing craniofacial region. The observed pattern suggested a potential involvement of BMP7 in epithelial-mesenchymal interactions and thus a direct role for this molecule in the development of ectodermal appendages (teeth, hair follicle, lachrymal and sweat glands, taste buds) and, furthermore, palatal formation. To correlate the expression to function we analyzed germline deleted conditional BMP7-deficient embryos for malformations. We found developmental defects in many craniofacial structures such as teeth, eyes, whiskers, hair follicles, salivary glands, and palate. These findings place BMP7 as a central mediator of epithelial-mesenchymal interactions that are necessary for the correct development of structures belonging to the orofacial complex.

Human Dental Pulp Stem Cells Hook into Biocoral Scaffold Forming an Engineered Biocomplex

The aim of this study was to evaluate the behavior of human Dental Pulp Stem Cells (DPSCs), as well as human osteoblasts, when challenged on a Biocoral scaffold, which is a porous natural hydroxyapatite. For this purpose, human DPSCs were seeded onto a three-dimensional (3D) Biocoral scaffold or on flask surface (control). Either normal or rotative (3D) cultures were performed. Scanning electron microscopic analyses, at 8, 24 and 48 h of culture showed that cells did not adhere on the external surface, but moved into the cavities inside the Biocoral structure. After 7, 15 and 30 days of culture, morphological and molecular analyses suggested that the Biocoral scaffold leads DPSCs to hook into the cavities where these cells quickly start to secrete the extra cellular matrix (ECM) and differentiate into osteoblasts. Control human osteoblasts also moved into the internal cavities where they secreted the ECM. Histological sections revealed a diffuse bone formation inside the Biocoral samples seeded with DPSCs or human osteoblasts, where the original scaffold and the new secreted biomaterial were completely integrated and cells were found within the remaining cavities. In addition, RT-PCR analyses showed a significant increase of osteoblast-related gene expression and, above all, of those genes highly expressed in mineralized tissues, including osteocalcin, OPN and BSP. Furthermore, the effects on the interaction between osteogenesis and angiogenesis were observed and substantiated by ELISA assays. Taken together, our results provide clear evidence that DPSCs differentiated into osteoblasts, forming a biocomplex made of Biocoral, ECM and differentiated cells.

Future dentistry: cell therapy meets tooth and periodontal repair and regeneration

•  Introduction ‐  The need for tooth tissue engineering ‐  Cellular components and development of tooth ‐  Bases for tooth tissue engineering ‐  The patient as a recipient and as the source of cells ‐  Dental stem cell based tissue engineering ‐  Dental pulp stem cells (DPSCs) ‐  The clinical application of DPSCs in regeneration of the pulp/dentin complex ‐  Periodontal tissues as a source and niche for stem cells ‐  Past, current and future approaches in periodontal regeneration ‐  Molecular mechanisms and factors regulating regeneration of periodontal tissues ‐  Scaffolding and material science •  Conclusions