Yuki Hayashi

Stimulation of angiogenesis, neurogenesis and regeneration by side population cells from dental pulp

Mesenchymal stem cells (MSCs) have been used for cell therapy in various experimental disease models. However, the regenerative potential of MSCs from different tissue sources and the influence of the tissue niche have not been investigated. In this study, we compared the regenerative potential of dental pulp, bone marrow and adipose tissue-derived CD31(-) side population (SP) cells isolated from an individual porcine source. Pulp CD31(-) SP cells expressed the highest levels of angiogenic/neurotrophic factors and had the highest migration activity. Conditioned medium from pulp CD31(-) SP cells produced potent anti-apoptotic activity and neurite outgrowth, compared to those from bone marrow and adipose CD31(-) SP cells. Transplantation of pulp CD31(-) SP cells in a mouse hindlimb ischemia model produced higher blood flow and capillary density than transplantation of bone marrow and adipose CD31(-) SP cells. Motor function recovery and infarct size reduction were greater with pulp CD31(-) SP cells. Pulp CD31(-) SP cells induced maximal angiogenesis, neurogenesis and pulp regeneration in ectopic transplantation models compared to other tissue sources. These results demonstrate that pulp stem cells have higher angiogenic, neurogenic and regenerative potential and may therefore be superior to bone marrow and adipose stem cells for cell therapy.

The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells with high regenerative potential.

Human dental pulp stem cells (DPSCs) contain subsets of progenitor/stem cells with high angiogenic, neurogenic and regenerative potential useful for cell therapy. It is essential to develop a safe and efficacious method to isolate the clinical-grade DPSCs subsets from a small amount of pulp tissue without using conventional flow cytometry. Thus, a method for isolation of DPSCs subsets based on their migratory response to optimized concentration of 100 ng/ml of granulocyte-colony stimulating factor (G-CSF) was determined in this study. The DPSCs mobilized by G-CSF (MDPSCs) were enriched for CD105, C-X-C chemokine receptor type 4 (CXCR-4) and G-CSF receptor (G-CSFR) positive cells, demonstrating stem cell properties including high proliferation rate and stability. The absence of abnormalities/aberrations in karyotype and lack of tumor formation after transplantation in an immunodeficient mouse were demonstrated. The conditioned medium of MDPSCs exhibited anti-apoptotic activity, enhanced migration and immunomodulatory properties. Furthermore, transplantation of MDPSCs accelerated vasculogenesis in an ischemic hindlimb model and augmented regenerated pulp tissue in an ectopic tooth root model compared to that of colony-derived DPSCs, indicating higher regenerative potential of MDPSCs. In conclusion, this isolation method for DPSCs subsets is safe and efficacious, having utility for potential clinical applications to autologous cell transplantation.

Isolation of a Stable Subpopulation of Mobilized Dental Pulp Stem Cells (MDPSCs) with High Proliferation, Migration, and Regeneration Potential Is Independent of Age

Insights into the understanding of the influence of the age of MSCs on their cellular responses and regenerative potential are critical for stem cell therapy in the clinic. We have isolated dental pulp stem cells (DPSCs) subsets based on their migratory response to granulocyte-colony stimulating factor (G-CSF) (MDPSCs) from young and aged donors. The aged MDPSCs were efficiently enriched in stem cells, expressing high levels of trophic factors with high proliferation, migration and anti-apoptotic effects compared to young MDPSCs. In contrast, significant differences in those properties were detected between aged and young colony-derived DPSCs. Unlike DPSCs, MDPSCs showed a small age-dependent increase in senescence-associated β-galactosidase (SA-β-gal) production and senescence markers including p16, p21, Interleukin (IL)-1β, -6, -8, and Groα in long-term culture. There was no difference between aged and young MDPSCs in telomerase activity. The regenerative potential of aged MDPSCs was similar to that of young MDPSCs in an ischemic hindlimb model and an ectopic tooth root model. These results demonstrated that the stem cell properties and the high regenerative potential of MDPSCs are independent of age, demonstrating an immense utility for clinical applications by autologous cell transplantation in dental pulp regeneration and ischemic diseases.

CXCL14 and MCP1 are potent trophic factors associated with cell migration and angiogenesis leading to higher regenerative potential of dental pulp side population cells

The release of trophic factors from mesenchymal stem cells (MSCs) is critical for tissue regeneration. A systematic investigation of the regenerative potential of trophic factors from different MSCs, however, has not been performed. Thus, in the present study, the regenerative potential of conditioned medium (CM) from dental pulp, bone marrow, and adipose tissue-derived CD31− side population (SP) cells from an individual source was compared in an ectopic tooth transplantation model. The tooth root transplantation in an ectopic site model was used for investigation of the regenerative potential and trophic effects in vivo. Either pulp CD31− SP cell populations (1×106 cells) at the third to fourth passage or 5 μg/ml of CM from dental pulp, bone marrow, and adipose stem cells from four different individuals were injected into the root with collagen TE. Each root was transplanted subcutaneously in 5-week-old severe combined immunodeficiency mice. Each root with surrounding tissue was harvested for histology on days 7, 21, and 28 and for Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis on day 28. Furthermore, the trophic factors responsible for the regenerative potential were identified as the upregulated genes present in pulp CD31− SP cells when compared with the genes in both bone marrow and adipose CD31− SP cells by using microarray analysis, real-time RT-PCR, and Western blot analysis. Transplantation of pulp CM yielded increased volume of pulp regeneration, more bromodeoxyuridine (BrdU)-positive migrated cells, and fewer caspase 3-positive cells in the regenerated pulp compared with the others. Pulp CM also demonstrated significantly increased cell migration, anti-apoptosis, and angiogenesis in C2C12 cells. Higher expression of CXCL14 and MCP1 in pulp SP cells suggested candidate trophic factors. The stimulatory effects on both migration and angiogenesis of CXCL14 and MCP1 were demonstrated in vitro. In the regenerated tissue, BrdU-positive migrated cells expressed CXCR4 and CCR2, receptors for CXCL14 and MCP1, respectively. The higher regenerative potential of pulp SP cells may be due to potent trophic factors, including CXCL14 and MCP1, which promote migration and angiogenesis.

Similar in vitro effects and pulp regeneration in ectopic tooth transplantation by basic fibroblast growth factor and granulocyte-colony stimulating factor.

OBJECTIVES Granulocyte-colony stimulating factor (G-CSF) has been shown to have combinatorial trophic effects with dental pulp stem cells for pulp regeneration. The aim of this investigation is to examine the effects of basic fibroblast growth factor (bFGF) in vitro and in vivo compared with those of G-CSF and to assess the potential utility of bFGF as an alternative to G-CSF for pulp regeneration. MATERIALS AND METHODS Five different types of cells were examined in the in vitro effects of bFGF on cell migration, proliferation, anti-apoptosis, neurite outgrowth, angiogenesis, and odontogenesis compared with those of G-CSF. The in vivo regenerative potential of pulp tissue including vasculogenesis and odontoblastic differentiation was also compared using an ectopic tooth transplantation model. RESULTS Basic fibroblast growth factor was similar to G-CSF in high migration, proliferation and anti-apoptotic effects and angiogenic and neurite outgrowth stimulatory activities in vitro. There was no significant difference between bFGF and G-CSF in the regenerative potential in vivo. CONCLUSIONS The potential utility of bFGF for pulp regeneration is demonstrated as a homing/migration factor similar to the influence of G-CSF.

Trophic Effects and Regenerative Potential of Mobilized Mesenchymal Stem Cells from Bone Marrow and Adipose Tissue as Alternative Cell Sources for Pulp/Dentin Regeneration:

Dental pulp stem cell (DPSC) subsets mobilized by granulocyte-colony-stimulating factor (G-CSF) are safe and efficacious for complete pulp regeneration. The supply of autologous pulp tissue, however, is very limited in the aged. Therefore, alternative sources of mesenchymal stem/progenitor cells (MSCs) are needed for the cell therapy. In this study, DPSCs, bone marrow (BM), and adipose tissue (AD)-derived stem cells of the same individual dog were isolated using G-CSF-induced mobilization (MDPSCs, MBMSCs, and MADSCs). The positive rates of CXCR4 and G-CSFR in MDPSCs were similar to MADSCs and were significantly higher than those in MBMSCs. Trophic effects of MDPSCs on angiogenesis, neurite extension, migration, and anti-apoptosis were higher than those of MBMSCs and MADSCs. Pulp-like loose connective tissues were regenerated in all three MSC transplantations. Significantly higher volume of regenerated pulp and higher density of vascularization and innervation were observed in response to MDPSCs compared to MBMSC and MADSC transplantation. Collagenous matrix containing dentin sialophosphoprotein (DSPP)-positive odontoblast-like cells was the highest in MBMSCs and significantly higher in MADSCs compared to MDPSCs. MBMSCs and MADSCs, therefore, have potential for pulp regeneration, although the volume of regenerated pulp tissue, angiogenesis, and reinnervation, were less. Thus, in conclusion, an alternative cell source for dental pulp/dentin regeneration are stem cells from BM and AD tissue.

Trophic Effects of Dental Pulp Stem Cells on Schwann Cells in Peripheral Nerve Regeneration

Recently, mesenchymal stem cells have demonstrated a potential for neurotrophy and neurodifferentiation. We have recently isolated mobilized dental pulp stem cells (MDPSCs) using granulocyte-colony stimulating factor (G-CSF) gradient, which has high neurotrophic/angiogenic potential. The aim of this study is to investigate the effects of MDPSC transplantation on peripheral nerve regeneration. Effects of MDPSC transplantation were examined in a rat sciatic nerve defect model and compared with autografts and control conduits containing collagen scaffold. Effects of conditioned medium of MDPSCs were also evaluated in vitro. Transplantation of MDPSCs in the defect demonstrated regeneration of myelinated fibers, whose axons were significantly higher in density compared with those in autografts and control conduits only. Enhanced revascularization was also observed in the MDPSC transplants. The MDPSCs did not directly differentiate into Schwann cell phenotype; localization of these cells near Schwann cells induced several neurotrophic factors. Immunofluorescence labeling demonstrated reduced apoptosis and increased proliferation in resident Schwann cells in the MDPSC transplant compared with control conduits. These trophic effects of MDPSCs on proliferation, migration, and antiapoptosis in Schwann cells were further elucidated in vitro. The results demonstrate that MDPSCs promote axon regeneration through trophic functions, acting on Schwann cells, and promoting angiogenesis.

EDTA soluble chemical components and the conditioned medium from mobilized dental pulp stem cells contain an inductive microenvironment, promoting cell proliferation, migration, and odontoblastic differentiation.

BackgroundThe critical challenge in tissue engineering is to establish an optimal combination of stem cells, signaling morphogenetic molecules, and extracellular matrix scaffold/microenvironment. The extracellular matrix components of teeth may be reconstituted as an inductive microenvironment in an ectopic tooth transplantation bioassay. Thus, the isolation and identification of the chemical components of the inductive microenvironment in pulp/dentin regeneration will accelerate progress towards the goal of tissue engineering of the tooth.MethodsThe teeth demineralized in 0.6 M hydrochloric acid were sequentially extracted by 4.0 M guanidine hydrochloride (GdnHCl), pH 7.4, and 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.4. The extracted teeth were transplanted into an ectopic site in severe combined immunodeficiency (SCID) mice with mobilized dental pulp stem cells (MDPSCs). The unextracted tooth served as a positive control. Furthermore, the soluble components for the inductive microenvironment, the GdnHCl extracts, or the EDTA extracts together with or without MDPSC conditioned medium (CM) were reconstituted systematically with autoclaved teeth in which the chemical components were completely inactivated and only the physical microenvironment was preserved. Their pulp/dentin regenerative potential and angiogenic potential were compared 28 days after ectopic tooth transplantation by histomorphometry and real-time RT-PCR analysis.ResultsExpression of an odontoblastic marker, enamelysin, and a pulp marker, thyrotropin-releasing hormone degrading enzyme (TRH-DE), was lower, and expression of a periodontal cell marker, anti-asporin/periodontal ligament-associated protein 1 (PLAP-1), was higher in the transplant of the EDTA-extracted teeth compared with the GdnHCl-extracted teeth. The autoclaved teeth reconstituted with the GdnHCl extracts or the EDTA extracts have weak regenerative potential and minimal angiogenic potential, and the CM significantly increased this potential. Combinatorial effects of the EDTA extracts and the CM on pulp/dentin regeneration were demonstrated in vivo, consistent with their in-vitro effects on enhanced proliferation, migration, and odontoblastic differentiation.ConclusionsThe EDTA-extracted teeth demonstrated significantly lower pulp/dentin regenerative potential compared with the GdnHCl-extracted teeth. The EDTA soluble chemical components when reconstituted with the physical structure of autoclaved teeth serve as an inductive microenvironment for pulp/dentin regeneration, promoting cell proliferation, migration, and odontoblastic differentiation.

Enhanced regeneration potential of mobilized dental pulp stem cells from immature teeth

OBJECTIVES We have previously demonstrated that dental pulp stem cells (DPSCs) isolated from mature teeth by granulocyte colony-stimulating factor (G-CSF)-induced mobilization method can enhance angiogenesis/vasculogenesis and improve pulp regeneration when compared with colony-derived DPSCs. However, the efficacy of this method in immature teeth with root-formative stage has never been investigated. Therefore, the aim of this study was to examine the stemness, biological characteristics, and regeneration potential in mobilized DPSCs compared with colony-derived DPSCs from immature teeth. MATERIALS AND METHODS Mobilized DPSCs isolated from immature teeth were compared to colony-derived DPSCs using methods including flow cytometry, migration assays, mRNA expression of angiogenic/neurotrophic factor, and induced differentiation assays. They were also compared in trophic effects of the secretome. Regeneration potential was further compared in an ectopic tooth transplantation model. RESULTS Mobilized DPSCs had higher migration ability and expressed more angiogenic/neurotrophic factors than DPSCs. The mobilized DPSC secretome produced a higher stimulatory effect on migration, immunomodulation, anti-apoptosis, endothelial differentiation, and neurite extension. In addition, vascularization and pulp regeneration potential were higher in mobilized DPSCs than in DPSCs. CONCLUSIONS G-CSF-induced mobilization method enhances regeneration potential of colony-derived DPSCs from immature teeth.