Takayoshi Yamaza

Mesenchymal Stem Cell-Mediated Functional Tooth Regeneration in Swine

Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.

Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study.

Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental pulp stem cells or DPSCs) and deciduous teeth (stem cells from human exfoliated deciduous teeth). We recently discovered another type of MSCs in the apical papilla of human immature permanent teeth termed stem cells from the apical papilla (SCAP). Here, we further characterized the apical papilla tissue and stem cell properties of SCAP using histologic, immunohistochemical, and immunocytofluorescent analyses. We found that the apical papilla is distinctive to the pulp in terms of containing less cellular and vascular components than those in the pulp. Cells in the apical papilla proliferated 2- to 3-fold greater than those in the pulp in organ cultures. Both SCAP and DPSCs were as potent in osteo/dentinogenic differentiation as MSCs from bone marrows, whereas they were weaker in adipogenic potential. The immunophenotype of SCAP is similar to that of DPSCs on the osteo/dentinogenic and growth factor receptor gene profiles. Double-staining experiments showed that STRO-1 coexpressed with dentinogenic markers such as bone sialophosphoprotein, osteocalcin, and growth factors FGFR1 and TGFbetaRI in cultured SCAP. Additionally, SCAP express a wide variety of neurogenic markers such as nestin and neurofilament M upon stimulation with a neurogenic medium. We conclude that SCAP are similar to DPSCs but a distinct source of potent dental stem/progenitor cells. Their implications in root development and apexogenesis are discussed.

Mesenchymal Stem Cell Transplantation Reverses Multiorgan Dysfunction in Systemic Lupus Erythematosus Mice and Humans

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease that, despite the advances in immunosuppressive medical therapies, remains potentially fatal in some patients, especially in treatment‐refractory patients. Here, we reported that impairment of bone marrow mesenchymal stem cells (BMMSCs) and their associated osteoblastic niche deficiency contribute in part to the pathogenesis of SLE‐like disease in MRL/lpr mice. Interestingly, allogenic BMMSC transplantation (MSCT) is capable of reconstructing the bone marrow osteoblastic niche and more effectively reverses multiorgan dysfunction when compared with medical immunosuppression with cyclophosphamide (CTX). At the cellular level, MSCT, not CTX treatment, was capable to induce osteoblastic niche reconstruction, possibly contributing to the recovery of regulatory T‐cells and reestablishment of the immune homeostasis. On the basis of the promising clinical outcomes in SLE mice, we treated four CTX/glucocorticoid treatment‐refractory SLE patients using allogenic MSCT and showed a stable 12–18 months disease remission in all treated patients. The patients benefited an amelioration of disease activity, improvement in serologic markers and renal function. These early evidences suggest that allogenic MSCT may be a feasible and safe salvage therapy in refractory SLE patients. STEM CELLS 2009;27:1421–1432

Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis

Systemic infusion of bone marrow mesenchymal stem cells (BMMSCs) yields therapeutic benefit for a variety of autoimmune diseases, but the underlying mechanisms are poorly understood. Here we show that in mice systemic infusion of BMMSCs induced transient T cell apoptosis via the FAS ligand (FASL)-dependent FAS pathway and could ameliorate disease phenotypes in fibrillin-1 mutated systemic sclerosis (SS) and dextran-sulfate-sodium-induced experimental colitis. FASL(-/-) BMMSCs did not induce T cell apoptosis in recipients, and could not ameliorate SS and colitis. Mechanistic analysis revealed that FAS-regulated monocyte chemotactic protein 1 (MCP-1) secretion by BMMSCs recruited T cells for FASL-mediated apoptosis. The apoptotic T cells subsequently triggered macrophages to produce high levels of TGFβ, which in turn led to the upregulation of CD4(+)CD25(+)Foxp3(+) regulatory T cells and, ultimately, immune tolerance. These data therefore demonstrate a previously unrecognized mechanism underlying BMMSC-based immunotherapy involving coupling via FAS/FASL to induce T cell apoptosis.

Immunomodulatory properties of stem cells from human exfoliated deciduous teeth

IntroductionStem cells from human exfoliated deciduous teeth (SHED) have been identified as a population of postnatal stem cells capable of differentiating into osteogenic and odontogenic cells, adipogenic cells, and neural cells. Herein we have characterized mesenchymal stem cell properties of SHED in comparison to human bone marrow mesenchymal stem cells (BMMSCs).MethodsWe used in vitro stem cell analysis approaches, including flow cytometry, inductive differentiation, telomerase activity, and Western blot analysis to assess multipotent differentiation of SHED and in vivo implantation to assess tissue regeneration of SHED. In addition, we utilized systemic SHED transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice.ResultsWe found that SHED are capable of differentiating into osteogenic and adipogenic cells, expressing mesenchymal surface molecules (STRO-1, CD146, SSEA4, CD73, CD105, and CD166), and activating multiple signaling pathways, including TGFβ, ERK, Akt, Wnt, and PDGF. Recently, BMMSCs were shown to possess an immunomodulatory function that leads to successful therapies for immune diseases. We examined the immunomodulatory properties of SHED in comparison to BMMSCs and found that SHED had significant effects on inhibiting T helper 17 (Th17) cells in vitro. Moreover, we found that SHED transplantation is capable of effectively reversing SLE-associated disorders in MRL/lpr mice. At the cellular level, SHED transplantation elevated the ratio of regulatory T cells (Tregs) via Th17 cells.ConclusionsThese data suggest that SHED are an accessible and feasible mesenchymal stem cell source for treating immune disorders like SLE.

BCOR regulates mesenchymal stem cell function by epigenetic mechanisms

The BCL-6 co-repressor (BCOR) represses gene transcription by interacting with BCL-6 (Refs 1, 2). BCOR mutation is responsible for oculo-facio-cardio-dental (OFCD) syndrome, which is characterized by canine teeth with extremely long roots, congenital cataracts, craniofacial defects and congenital heart disease. Here we show that BCOR mutation increased the osteo-dentinogenic potential of mesenchymal stem cells (MSCs) isolated from a patient with OFCD, providing a molecular explanation for abnormal root growth. AP-2α was identified as a repressive target of BCOR, and BCOR mutation resulted in abnormal activation of AP-2α. Gain- and loss-of-function assays suggest that AP-2α is a key factor that mediates the increased osteo-dentinogenic capacity of MSCs. Moreover, we found that BCOR maintained tissue homeostasis and gene silencing through epigenetic mechanisms. BCOR mutation increased histone H3K4 and H3K36 methylation in MSCs, thereby reactivating transcription of silenced target genes. By studying a rare human genetic disease, we have unravelled an epigenetic mechanism for control of human adult stem cell function.

Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential

BACKGROUND Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). AIMS To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. MATERIALS & METHODS DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. RESULTS The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 + or - 2.9) was lower than that of DPSCs-NPs (58.9 + or - 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. CONCLUSION DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo.

Pharmacologic Stem Cell Based Intervention as a New Approach to Osteoporosis Treatment in Rodents

Background Osteoporosis is the most prevalent skeletal disorder, characterized by a low bone mineral density (BMD) and bone structural deterioration, leading to bone fragility fractures. Accelerated bone resorption by osteoclasts has been established as a principal mechanism in osteoporosis. However, recent experimental evidences suggest that inappropriate apoptosis of osteoblasts/osteocytes accounts for, at least in part, the imbalance in bone remodeling as occurs in osteoporosis. The aim of this study is to examine whether aspirin, which has been reported as an effective drug improving bone mineral density in human epidemiology studies, regulates the balance between bone resorption and bone formation at stem cell levels. Methods and Findings We found that T cell-mediated bone marrow mesenchymal stem cell (BMMSC) impairment plays a crucial role in ovariectomized-induced osteoporosis. Ex vivo mechanistic studies revealed that T cell-mediated BMMSC impairment was mainly attributed to the apoptosis of BMMSCs via the Fas/Fas ligand pathway. To explore potential of using pharmacologic stem cell based intervention as an approach for osteoporosis treatment, we selected ovariectomy (OVX)-induced ostoeporosis mouse model to examine feasibility and mechanism of aspirin-mediated therapy for osteoporosis. We found that aspirin can inhibit T cell activation and Fas ligand induced BMMSC apoptosis in vitro. Further, we revealed that aspirin increases osteogenesis of BMMSCs by aiming at telomerase activity and inhibits osteoclast activity in OVX mice, leading to ameliorating bone density. Conclusion Our findings have revealed a novel osteoporosis mechanism in which activated T cells induce BMMSC apoptosis via Fas/Fas ligand pathway and suggested that pharmacologic stem cell based intervention by aspirin may be a new alternative in osteoporosis treatment including activated osteoblasts and inhibited osteoclasts.

Study of immunoelectron microscopic localization of cathepsin K in osteoclasts and other bone cells in the mouse femur

The localization of cathepsin K protein in mouse osteoclasts was examined by immunolight and immunoelectron microscopy using the avidin-biotin-peroxidase complex method with anti-cathepsin K (mouse) antibody. With light microscopy, a strong immunoreaction for cathepsin K was found extracellularly along the bone and cartilage resorption lacunae and detected intracellularly in vesicles, granules, and vacuoles throughout the cytoplasm of multinuclear osteoclasts and chondroclasts attached to the surface of the bone or cartilage. Mononuclear cells, probably preosteoclasts, some distance from the bone also contained a few cathepsin K-positive vesicles and granules. Cathepsin K was sometimes found in the cisternal spaces of the rough endoplasmic reticulum and vesicles of the Golgi apparatus with electron microscopy of the basolateral region of the osteoclasts. Cathepsin K-positive vesicles and granules as lysosomal compartments were present in various stages of fusion with vacuoles as endosomal compartments that contained fragmented cathepsin K-negative fibril-like structures. Some of the vacuoles (endolysosomes), which seemed to be formed by this process of fusion, contained cathepsin K-positive vesicles and fibril-like structures that did not show the regular cross striation of type I collagen fibrils. In the apical region of the osteoclasts, cathepsin K-positive vesicles and pits had already fused with or were in the process of fusing with the ampullar extracellular spaces. There were large deposits of cathepsin K on fragmented fibril-like structures without regular cross striation in the extracellular spaces, as well as on and between the cytoplasmic processes of the ruffled border. There were also extensive deposits of cathepsin K on the type I collagen fibrils with cross striation in the bone resorption lacunae. Osteoblasts and osteocytes were negative for cathepsin K. In the immunocytochemical controls, no immunoreaction was found in the osteoclasts or preosteoclasts, or on the collagen fibrils in the resorption lacunae. The results indicate that cathepsin K is produced in mature osteoclasts attached to the bone and secreted into the bone resorption lacunae. The findings suggest that cathepsin K participates in the extracellular degradation of collagen fibrils in the resorption lacunae and in the subsequent degradation of the fragmented fibrils in the endolysosomes. It is also suggested that cathepsin K degrades the organic cartilage matrix.

Human Hertwig’s Epithelial Root Sheath Cells Play Crucial Roles in Cementum Formation

Hertwig’s epithelial root sheath (HERS) cells are a unique population of epithelial cells in the periodontal ligament compartment. To date, their functional role has not been fully elucidated. Our hypothesis was that HERS cells may be involved in regulating differentiation of periodontal ligament stem cells (PDLSCs) and forming cementum in vivo. In this study, we found that HERS cells may be capable of promoting PDLSC differentiation and undergoing epithelial-mesenchymal transition in vitro. Immunohistochemical staining, Western blot analysis, a transwell co-culture system, and in vivo transplantation were used to characterize the interplay between HERS cells and PDLSCs, as well as the epithelial-mesenchymal transition (EMT) of HERS cells. TGFβ1 was capable of inducing the epithelial-mesenchymal transition of HERS cells through activating the PI3K/AKT pathway. Furthermore, HERS cells were able to form cementum-like tissue when transplanted into immunocompromised mice. Abbreviations: bone marrow mesenchymal stem cell, BMMSC; bone sialoprotein, BSP; hydroxyapatite/tricalcium phosphate, HA/TCP; Hertwig’s epithelial root sheath, HERS; osteocalcin, OCN; periodontal ligament, PDL; periodontal ligament stem cell, PDLSC; phosphatidylinositol 3-kinase, PI3K.