Published papers

This study investigated a case of spindle cell carcinoma (SpCC) in tongue pathological lesions. The patient experienced a local recurrence and distant metastasis after surgical intervention. Although standard chemotherapy was administered, a granulomatous mass continued to develop. This aggressive growth led to survival of the tumor. Secondary debulking surgery was performed to improve the patient's quality of life at the request of the patient. Using a tissue sample derived from the secondary debulking surgery, we performed an analysis of the tumor's cell surface antigens, differentiation potential, metastatic ability, and inhibition potential by anticancer reagents. In vitro analysis revealed that the cell population grown under adherent culture conditions expressed the mesenchymal stem cell (MSC) markers CD73, CD90, and CD105. The cell line established from this SpCC contained colony-forming unit fibroblasts (CFU-Fs) and exhibited multipotent differentiation into several mesenchymal lineages, including bone, cartilage, and fat. The SpCC cells also displayed vigorous mobilization. These characteristics suggested that they had the differentiation potential of mesenchymal cells, especially MSCs, rather than that of epithelial cells. The surgical specimen analyzed in this study resisted the molecular target reagent cetuximab, which is an epidermal growth factor receptor inhibitor. This clinical insight revealed that chemotherapy-resistant SpCC cells have different characteristics compared to most other cancer cells, which are sensitive to cetuximab. Our cell death assay revealed that SpCC cell death was induced by the anticancer drug imatinib, which is known to inhibit protein tyrosine kinase activity of ABL, platelet-derived growth factor receptor alpha (PDGFRalpha), and KIT. Here, we report recurrent SpCC with characteristics of MSCs and potential for treatment with imatinib.

Behcet's disease is a systemic disorder of unknown etiology. It involves multiple organ systems and is characterized by recurring episodes of oral ulcers as well as ocular, genital, and skin lesions. Oral ulcers can affect tooth brushing and impair proper oral hygiene. As a result, a dental biofilm accumulates, and the condition of the teeth and periodontal tissue deteriorates. The aim of this case report is to highlight the efficacy of periodontal treatment for patients with Behcet's disease. A 51-year-old man with Behcet's disease presented with generalized severe periodontitis. After basic treatment of the periodontal tissues, periodontal surgery was performed at several sites with bony defects. However, the patient developed severe stomatitis in the oral mucosa and gingiva after periodontal surgery. Administration of the antimicrobial agent cefdinir had little effect on recovery; however, subsequent administration of sitafloxacin resulted in significant improvement of the stomatitis. This case demonstrates that periodontal therapy is very useful for alleviating the oral signs and symptoms of Behcet's disease. Systemic antibiotic treatment with sitafloxacin (but not cefdinir) and mechanical debridement were effective in preventing the recurrence of aphthous ulcer outbreaks after periodontal surgery.

PURPOSE: This study aimed to clarify the mechanisms and assess the characteristics of the chronic graft-versus-host disease (cGVHD) fibrosis in the lacrimal gland (LG) of mice. METHODS: Histopathology of LG tissues was examined by immunohistochemistry and electron microscopy. Cultured fibroblasts derived from the LG were analyzed by phase-contrast microscopy, immunocytochemistry, flow cytometry, proliferation assay, and invasion and migration assays. RESULTS: Cultured murine LG fibroblasts in cGVHD were spindle-shaped and relatively small, whereas those from syngeneic controls were polygon-shaped and relatively large. Flow cytometric analysis showed that the LG fibroblasts in cGVHD had elevated HSP47 levels. The LG fibroblasts in cGVHD also showed increased expression of major histocompatibility complex class II. Furthermore, the proportion of Sca-1PDGFR-alpha cells among the LG fibroblasts in cGVHD was considerably increased compared with controls. Cell counting kit-8 assays demonstrated that the LG fibroblasts in cGVHD were highly proliferative, and cell invasion assays indicated that they were highly invasive with high migration ability. CONCLUSIONS: LG fibroblasts in cGVHD can be aberrantly activated, thereby eliciting fibrosis by producing excessive extracellular matrix, leading to LG dysfunction in mice.

Effective regenerative treatments for periodontal tissue defects have recently been demonstrated using mesenchymal stromal/stem cells (MSCs). Furthermore, current bioengineering techniques have enabled de novo fabrication of tooth-perio dental units in mice. These cutting-edge technologies are expected to address unmet needs within regenerative dentistry. However, to achieve efficient and stable treatment outcomes, preparation of an appropriate stem cell source is essential. Many researchers are investigating the use of adult stem cells for regenerative dentistry; bone marrow-derived MSCs (BM-MSCs) are particularly promising and presently used clinically. However, current BM-MSC isolation techniques result in a heterogeneous, non-reproducible cell population because of a lack of identified distinct BM-MSC surface markers. Recently, specific subsets of cell surface markers for BM-MSCs have been reported in mice (PDGFRalpha(+) and Sca-1(+)) and humans (LNGFR(+), THY-1(+) and VCAM-1(+)), facilitating the isolation of unique enriched BM-MSCs (so-called "purified MSCs"). Notably, the enriched BM-MSC population contains neural crest-derived cells, which can differentiate into cells of neural crest- and mesenchymal lineages. In this review, characteristics of the enriched BM-MSCs are outlined with a focus on their potential application within future regenerative dentistry.

Dental pulp stem cells/progenitor cells (DPSCs) can be easily obtained and can have excellent proliferative and mineralization potentials. Therefore, many studies have investigated the isolation and bone formation of DPSCs. In most previous reports, human DPSCs were traditionally isolated by exploiting their ability to adhere to plastic tissue culture dishes. DPSCs isolated by plastic adherence are frequently contaminated by other cells, which limits the ability to investigate their basic biology and regenerative properties. Additionally, the proliferative and osteogenic potentials vary depending on the isolated cells. It is very difficult to obtain cells of a sufficient quality to elicit the required effect upon transplantation. Considering clinical applications, stem cells used for regenerative medicine need to be purified in order to increase the efficiency of bone regeneration, and a stable supply of these cells must be generated. Here, we review the purification of DPSCs and studies of cranio-maxillofacial bone regeneration using these cells. Additionally, we introduce the prospective isolation of DPSCs using specific cell surface markers: low-affinity nerve growth factor and thymocyte antigen 1.

Craniofacial skeletal tissues are composed of tooth and bone, together with nerves and blood vessels. This composite material is mainly derived from neural crest cells (NCCs). The neural crest is transient embryonic tissue present during neural tube formation whose cells have high potential for migration and differentiation. Thus, NCCs are promising candidates for craniofacial tissue regeneration; however, the clinical application of NCCs is hindered by their limited accessibility. In contrast, mesenchymal stem cells (MSCs) are easily accessible in adults, have similar potential for self-renewal, and can differentiate into skeletal tissues, including bones and cartilage. Therefore, MSCs may represent good sources of stem cells for clinical use. MSCs are classically identified under adherent culture conditions, leading to contamination with other cell lineages. Previous studies have identified mouse- and human-specific MSC subsets using cell surface markers. Additionally, some studies have shown that a subset of MSCs is closely related to neural crest derivatives and endothelial cells. These MSCs may be promising candidates for regeneration of craniofacial tissues from the perspective of developmental fate. Here, we review the fundamental biology of MSCs in craniofacial research.

Mesenchymal stem/stromal cells (MSCs) reside in the bone marrow and maintain their stemness under hypoxic conditions. However, the mechanism underlying the effects of hypoxia on MSCs remains to be elucidated. This study attempted to uncover the signaling pathway of MSC proliferation. Under low-oxygen culture conditions, MSCs maintained their proliferation and differentiation abilities for a long term. The Notch2 receptor was up-regulated in MSCs under hypoxic conditions. Notch2-knockdown (Notch2-KD) MSCs lost their cellular proliferation ability and showed reduced gene expression of hypoxia-inducible transcription factor (HIF)-1alpha, HIF-2alpha, and c-Myc. Overexpression of the c-Myc gene in Notch2-KD MSCs allowed the cells to regain their proliferation capacity. These results suggested that Notch2 signaling is linked to c-Myc expression and plays a key role in the regulation of MSC proliferation. Our findings provide important knowledge for elucidating the self-replication competence of MSCs in the bone marrow microenvironment.

Mesenchymal stem cells (MSCs) are defined as non-hematopoietic, plastic-adherent, self-renewing cells that are capable of tri-lineage differentiation into bone, cartilage or fat in vitro. Thus, MSCs are promising candidates for cell-based medicine. However, classifications of MSCs have been defined retrospectively; moreover, this conventional criterion may be inaccurate due to contamination with other hematopoietic lineage cells. Human MSCs can be enriched by selection for LNGFR and THY-1, and this population may be analogous to murine PDGFRalpha(+)Sca-1(+) cells, which are developmentally derived from neural crest cells (NCCs). Murine NCCs were labeled by fluorescence, which provided definitive proof of neural crest lineage, however, technical considerations prevent the use of a similar approach to determine the origin of human LNGFR(+)THY-1(+) MSCs. To further clarify the origin of human MSCs, human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) were used in this study. Under culture conditions required for the induction of neural crest cells, human ESCs and iPSCs-derived cells highly expressed LNGFR and THY-1. These LNGFR(+)THY-1(+) neural crest-like cells, designated as LT-NCLCs, showed a strong potential to differentiate into both mesenchymal and neural crest lineages. LT-NCLCs proliferated to form colonies and actively migrated in response to serum concentration. Furthermore, we transplanted LT-NCLCs into chick embryos, and traced their potential for survival, migration and differentiation in the host environment. These results suggest that LNGFR(+)THY-1(+) cells identified following NCLC induction from ESCs/iPSCs shared similar potentials with multipotent MSCs.

Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies.

Fibrosis of organs is observed in systemic autoimmune disease. Using a scleroderma mouse, we show that transplantation of MHC compatible, minor antigen mismatched bone marrow stromal/stem cells (BMSCs) play a role in the pathogenesis of fibrosis. Removal of donor BMSCs rescued mice from disease. Freshly isolated PDGFRalpha(+) Sca-1(+) BMSCs expressed MHC class II following transplantation and activated host T cells. A decrease in FOXP3(+) CD25(+) Treg population was observed. T cells proliferated and secreted IL-6 when stimulated with mismatched BMSCs in vitro. Donor T cells were not involved in fibrosis because transplanting T cell-deficient RAG2 knock out mice bone marrow still caused disease. Once initially triggered by mismatched BMSCs, the autoimmune phenotype was not donor BMSC dependent as the phenotype was observed after effector T cells were adoptively transferred into naive syngeneic mice. Our data suggest that minor antigen mismatched BMSCs trigger systemic fibrosis in this autoimmune scleroderma model.

Repeated and dramatic pregnancy-induced uterine enlargement and remodeling throughout reproductive life suggests the existence of uterine smooth muscle stem/progenitor cells. The aim of this study was to isolate and characterize stem/progenitor-like cells from human myometrium through identification of specific surface markers. We here identify CD49f and CD34 as markers to permit selection of the stem/progenitor cell-like population from human myometrium and show that human CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells exhibit stem cell-like properties. These include side population phenotypes, an undifferentiated status, high colony-forming ability, multilineage differentiation into smooth muscle cells, osteoblasts, adipocytes, and chondrocytes, and in vivo myometrial tissue reconstitution following xenotransplantation. Furthermore, CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells proliferate under hypoxic conditions in vitro and, compared with the untreated nonpregnant myometrium, show greater expansion in the estrogen-treated nonpregnant myometrium and further in the pregnant myometrium in mice upon xenotransplantation. These results suggest that the newly identified myometrial stem/progenitor-like cells influenced by hypoxia and sex steroids may participate in pregnancy-induced uterine enlargement and remodeling, providing novel insights into human myometrial physiology.

Hematopoietic stem cells in the bone marrow have the capacity to both self-renew and to generate all cells of the hematopoietic system. The balance of these two activities is controlled by hematopoietic stem cell-intrinsic regulatory mechanisms as well as extrinsic signals from the microenvironment. Here we demonstrate that Meis1, a TALE family homeodomain transcription factor involved in numerous embryonic developmental processes, is selectively expressed in hematopoietic stem/progenitor cells. Conditional Meis1 knockout in adult hematopoietic cells resulted in a significant reduction in the hematopoietic stem/progenitor cells. Suppression of hematopoiesis by Meis1 deletion appears to be caused by impaired self-renewal activity and reduced cellular quiescence of hematopoietic stem/progenitor cells in a cell autonomous manner, resulting in stem cell exhaustion and defective long-term hematopoiesis. Meis1 deficiency down-regulated a subset of Pbx1-dependent hematopoietic stem cell signature genes, suggesting a functional link between them in the maintenance of hematopoietic stem/progenitor cells. These results show the importance of Meis1 in adult hematopoiesis.

Human mesenchymal stem cells (hMSCs), which conventionally are isolated based on their adherence to plastic, are heterogeneous and have poor growth and differentiation, limiting our ability to investigate their intrinsic characteristics. We report an improved prospective clonal isolation technique and reveal that the combination of three cell-surface markers (LNGFR, THY-1, and VCAM-1) allows for the selection of highly enriched clonogenic cells (one out of three isolated cells). Clonal characterization of LNGFR(+)THY-1(+) cells demonstrated cellular heterogeneity among the clones. Rapidly expanding clones (RECs) exhibited robust multilineage differentiation and self-renewal potency, whereas the other clones tended to acquire cellular senescence via P16INK4a and exhibited frequent genomic errors. Furthermore, RECs exhibited unique expression of VCAM-1 and higher cellular motility compared with the other clones. The combination marker LNGFR(+)THY-1(+)VCAM-1(hi+) (LTV) can be used selectively to isolate the most potent and genetically stable MSCs.

マウス成体組織、間葉系幹細胞(MSC)および尻尾線維芽細胞からセンダイウイルスを用いてゲノム組込みのない人工多能性幹細胞(iPS細胞)を作成した。続いて、成体MSC由来のレトロウイルス誘導iPS細胞株および非組込みiPS細胞株から形成された二次ニューロスフェアに残存する未分化細胞を検出した。その結果、MSCからのみ組込みのないiPS細胞が得られた。しかし、非組込み法で得られたiPS細胞株の一部も未分化細胞を含んでいた。組込みのないiPS細胞のうち正しく分化しないものはフローサイトメトリーにて他のES/iPS細胞よりも高い側方散乱光(SSC)強度を示した。いくつかの体細胞由来iPS細胞は高いSSC強度を示し、これらの細胞は正常に分化しなかった。SSC強度の解析は個別のiPS細胞の評価に有効な手段である。

Platelet-derived growth factor receptor alpha (PDGFR-alpha) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-alpha(+)Sca-1(+) (PalphaS) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of PalphaS MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate PalphaS MSCs using flow cytometry. In brief, the tibia and femora are isolated and crushed using a pestle and mortar. The crushed bones are then chopped and incubated for 1 h at 37 degrees C in 20 ml of DMEM containing 0.2% (wt/vol) collagenase. The cell suspension is filtered before red blood cell lysis and incubated with the following antibodies: allophycocyanin (APC)-conjugated PDGFR-alpha, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45(+)Ter-119(+))-positive cells. Approximately 10,000 PalphaS MSCs may then be isolated per mouse. The total protocol takes ~7 h to complete.

OBJECTIVE: To investigate the antibacterial activity of povidone-iodine (PVP-I) on an artificial dual species biofilm of periodontal pathogens. DESIGN: Porphyromonas gingivalis or Fusobacterium nucleatum grown in broth culture was inoculated on polycarbonate membrane (PCM) tissue culture inserts. After incubation for 72 h, PVP-I solutions were applied to the biofilm for the time period ranging from 0.5 to 5 min. After addition of a deactivator, each PCM was removed and the biofilm on the PCM was serially diluted and plated on blood agar plates and cultured anaerobically for 7 days. Then viable bacteria were enumerated. RESULTS: In the dual species biofilm model, F. nucleatum showed an approximately 200-fold increase in viable counts when compared with mono-microbial biofilm. In dual species biofilm, PVP-I with concentration equal to or greater than 2% was required to significantly reduce P. gingivalis and F. nucleatum. When the contact time of PVP-I was increased to 1 min or greater, no difference in antibacterial activity of PVP-I was observed in any concentration. CONCLUSION: These results suggest that 30s application of 2% PVP-I would be effective in suppressing both P. gingivalis and F. nucleatum in dual-species biofilm, and this provides clinical implication for the control of subgingival biofilm.

BACKGROUND: Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by the forced expression of defined transcription factors. Although most somatic cells are capable of acquiring pluripotency with minimal gene transduction, the poor efficiency of cell reprogramming and the uneven quality of iPS cells are still important problems. In particular, the choice of cell type most suitable for inducing high-quality iPS cells remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here, we generated iPS cells from PDGFRalpha+ Sca-1+ (PalphaS) adult mouse mesenchymal stem cells (MSCs) and PDGFRalpha(-) Sca-1(-) osteo-progenitors (OP cells), and compared the induction efficiency and quality of individual iPS clones. MSCs had a higher reprogramming efficiency compared with OP cells and Tail Tip Fibroblasts (TTFs). The iPS cells induced from MSCs by Oct3/4, Sox2, and Klf4 appeared to be the closest equivalent to ES cells by DNA microarray gene profile and germline-transmission efficiency. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that a purified source of undifferentiated cells from adult tissue can produce high-quality iPS cells. In this context, prospectively enriched MSCs are a promising candidate for the efficient generation of high-quality iPS cells.

初期中胚葉特異的Mesp1-Cre/Floxed-EGFPをコードする成体トランスジェニックマウスの骨髄中に、沿軸中胚葉由来の間葉系幹細胞(MSC)を同定した。少量ではあるが有意な量のMesp1+細胞が成体骨髄に存在していた。骨髄中に検出されるEGFP+細胞の大部分は造血細胞画分に存在し、EGFP+MSCは骨細胞、脂肪細胞、軟骨細胞、神経細胞、グリア細胞および筋線維芽細胞に分化した。Mesp1-Cre/Floxed-EGFPマウス由来EGFP+MSCとEGFP-MSCとの間でin vitro特性の差はみられなかった。以上より、成体骨髄のMSCは複数の発生学的起源を持ち、MSCの一部はMesp1+初期沿軸中胚葉に由来すると考えられた。

We previously reported the beneficial effect of administering an anti-mouse IL-6 receptor antibody (MR16-1) immediately after spinal cord injury (SCI). The purpose of our present study was to clarify the mechanism underlying how MR16-1 improves motor function after SCI. Quantitative analyses of inflammatory cells using flow cytometry, and immunohistochemistry with bone marrow-chimeric mice generated by transplanting genetically marked purified hematopoietic stem cells, revealed that MR16-1 dramatically switched the central player in the post-traumatic inflammation, from hematogenous macrophages to resident microglia. This change was accompanied by alterations in the expression of relevant cytokines within the injured spinal cord; the expression of recruiting chemokines including CCL2, CCL5, and CXCL10 was decreased, while that of Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF), a known mitogen for microglia, was increased. We also showed that the resident microglia expressed higher levels of phagocytic markers than the hematogenous macrophages. Consistent with these findings, we observed significantly decreased tissue damage and reduced levels of myelin debris and Nogo-A, the axonal growth inhibitor, by MR16-1 treatment. Moreover, we observed increased axonal regeneration and/or sprouting in the MR16-1-treated mice. Our findings indicate that the functional improvement elicited by MR16-1 involves microglial functions, and provide new insights into the role of IL-6 signaling in the pathology of SCI.

オールトランスレチノイン酸(RA)が乳癌耐性蛋白質(Abcg2)を阻害することを発見した。トランスポーター阻害薬レセルピンと同様に、RAは新たな転写の必要なく、造血幹細胞(HSC)のAbcg2を阻害することによりHoechst色素排出を用量依存的に抑制した。side population細胞に対するRAとレセルピンの作用は可逆的であり、wash-outにより消失した。Abcg2を安定発現するNIH3T3-GFP細胞でも同様の結果が得られた。RA処理HSCおよびAbcg2欠損HSCはドキソルビシンに高い感受性を示した。以上より、HSCの生存、増殖および分化以外に、RAはAbcg2トランスポーター阻害物質として機能すると考えられた。