ラットBRONJ様モデルにおける幹細胞培養上清由来成⻑因⼦を用いた治療法の検討

Periodontal Tissue Regeneration with the stem cells cultured conditioned media.

Language：English   Publishing type：Research paper (scientific journal)  

Objectives: In this study, we investigated the diagnostic utility of submandibular gland (SMG) sonography and labial salivary gland (LSG) biopsy as a less invasive procedure for diagnosing IgG4-related dacryoadenitis and sialadenitis (IgG4-DS) Methods: Sixty-eight patients with suspected IgG4-DS by presenting swelling of elevated serum IgG (>1747 mg/dl) and/or swelling glands underwent SMG sonography, LSG biopsy and measurement for serum IgG4. SMG sonographic diagnosis was determined by the following characteristic changes; ‘hypoechoic areas of a nodal pattern with high vascularity’ and/or ‘hypoechoic areas of a reticular pattern in the superficial part’. Results: Thirty-one patients were diagnosed with IgG4-DS, 5 with IgG4-RD unaccompanied by lacrimal and salivary gland lesions, 28 with Sjögren’s syndrome, and 4 with malignant lymphoma. The sensitivity, specificity, and accuracy of SMG sonography and LSG biopsy were 100%, 83.8%, 91.2% and 64.5%, 73.8%, 75.0%, respectively. Moreover, those of SMG sonography and LSG biopsy combined with serum IgG4 concentration (>135 mg/dl) were 100%, 94.6%, 97.1% and 64.5%, 91.9%, 79.4%, respectively. Conclusion: LSG biopsy needs to be extremely careful to diagnose IgG4-DS because of its low sensitivity. SMG sonography is sufficient for the diagnosis of IgG4-DS, especially when combined with serologic analysis. Thus, SMG sonography could adapt to the diagnostic criteria of IgG4-DS as a non-invasive method.

DOI： 10.1080/14397595.2019.1576271

Language：English   Publishing type：Research paper (scientific journal)  

Human linkage studies suggest that craniofacial deformities result from either genetic mutations related to cholesterol metabolism or high-cholesterol maternal diets. However, little is known about the precise roles of intracellular cholesterol metabolism in the development of craniofacial bones, the majority of which are formed through intramembranous ossification. Here, we show that an altered cholesterol metabolic status results in abnormal osteogenesis through dysregulation of primary cilium formation during bone formation. We found that cholesterol metabolic aberrations, induced through disruption of either Dhcr7 (which encodes an enzyme involved in cholesterol synthesis) or Insig1 and Insig2 (which provide a negative feedback mechanism for cholesterol biosynthesis), result in osteoblast differentiation abnormalities. Notably, the primary cilia responsible for sensing extracellular cues were altered in number and length through dysregulated ciliary vesicle fusion in Dhcr7 and Insig1/2 mutant osteoblasts. As a consequence, WNT/β-catenin and hedgehog signaling activities were altered through dysregulated primary cilium formation. Strikingly, the normalization of defective cholesterol metabolism by simvastatin, a drug used in the treatment of cholesterol metabolic aberrations, rescued the abnormalities in both ciliogenesis and osteogenesis in vitro and in vivo. Thus, our results indicate that proper intracellular cholesterol status is crucial for primary cilium formation during skull formation and homeostasis.

DOI： 10.1038/s41413-019-0078-3

Language：English  

BACKGROUND: Cleft lip (CL), one of the most common congenital birth defects, shows considerable geographic and ethnic variation, with contribution of both genetic and environmental factors. Mouse genetic studies have identified several CL-associated genes. However, it remains elusive how these CL-associated genes are regulated and involved in CL. Environmental factors may regulate these genes at the post-transcriptional level through the regulation of non-coding microRNAs (miRNAs). In this study, we sought to identify miRNAs associated with CL in mice.
RESULTS: Through a systematic literature review and a Mouse Genome Informatics (MGI) database search, we identified 55 genes that were associated with CL in mice. Subsequent bioinformatic analysis of these genes predicted that a total of 33 miRNAs target multiple CL-associated genes, with 20 CL-associated genes being potentially regulated by multiple miRNAs. To experimentally validate miRNA function in cell proliferation, we conducted cell proliferation/viability assays for the selected five candidate miRNAs (miR-124-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p). Overexpression of miR-124-3p, but not of the others, inhibited cell proliferation through suppression of CL-associated genes in cultured mouse embryonic lip mesenchymal cells (MELM cells) isolated from the developing mouse lip region. By contrast, miR-124-3p knockdown had no effect on MELM cell proliferation. This miRNA-gene regulatory mechanism was mostly conserved in O9-1 cells, an established cranial neural crest cell line. Expression of miR-124-3p was low in the maxillary processes at E10.5, when lip mesenchymal cells proliferate, whereas it was greatly increased at later developmental stages, suggesting that miR-124-3p expression is suppressed during the proliferation phase in normal palate development.
CONCLUSIONS: Our findings indicate that upregulated miR-124-3p inhibits cell proliferation in cultured lip cells through suppression of CL-associated genes. These results will have a significant impact, not only on our knowledge about lip morphogenesis, but also on the development of clinical approaches for the diagnosis and prevention of CL.

DOI： 10.1186/s12864-019-6238-4

Language：English  

It has been long appreciated that sex hormone receptors are expressed in various non-gonadal organs. However, it remains unclear how sex hormones regulate the morphogenesis of these non-gonadal organs. To address this issue, we used a male mouse model of androgen-dependent salivary gland morphogenesis. Mice with excessive cholesterol synthesis in the salivary glands exhibited defects in the maturation of granular convoluted tubules (GCTs), which is regulated through sex hormone-dependent cascades. We found that excessive cholesterol synthesis resulted in autophagy failure specifically in the duct cells of salivary glands, followed by the accumulation of NRF2, a transcription factor known as one of the specific substrates for autophagy. The accumulated NRF2 suppressed the expression of Foxa1, which forms a transcriptional complex with the androgen receptor to regulate target genes. Taken together, our results indicate that cholesterol metabolism plays a crucial role in GCT differentiation through autophagy.

DOI： 10.1242/dev.178335

Language：English  

Objective: We previously reported that secretomes from human bone marrow-derived mesenchymal stem cells (MSC-CM) have a strong potential to accelerate bone regeneration. The most important initial step for bone regeneration is osteoprogenitor cell migration to bone defects. We hypothesized that MSC-CM enhance the migration of endogenous stem cells earlier to the local lesioned part. In this study, we investigated the potential of MSC-CM to induce in vivo early bone regeneration by accelerating cell migration in a rat calvarial bone defect model. Materials and methods: Cytokine array analysis was performed to assess the types of cytokines included in MSC-CM. Bone defects (5 mm in diameter) were created in the calvarial bones of rats, and the damaged areas were implanted with atelocollagen suspended in MSC-CM or phosphate buffered saline. After 2 and 4 weeks, radiographic and histological analyses were performed. Furthermore, rat mesenchymal stem cells (rMSCs) were labeled with the lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR), and the rats were photographed at various times after injection of the DiR-labeled rMSCs using in vivo imaging. Results: MSC-CM contained many factors with respect to cell migration and tissue regeneration. Bone regeneration in rat calvaria was observed earliest in the MSC-CM implantation group. Migration of the labeled rMSCs from the tail toward the calvaria, where MSC-CM was implanted, was observed during the first 24 h after injection in the MSC-CM implantation group using in vivo imaging. Immunohistochemistry also indicated early cell migration. Conclusion: MSC-CM enhanced the migration of endogenous stem cells facilitating earlier bone regeneration.

DOI： 10.1016/j.ajoms.2018.04.002

Language：English  

Recently, several studies have demonstrated that intravenous administration of mesenchymal stem cells (MSCs) improve medication-related osteonecrosis of the jaw (MRONJ), and paracrine effects of secretomes from MSCs have been hypothesized as the primary contributors. These secretomes in conditioned media from human MSCs (MSC-CM) were previously demonstrated to promote bone and tissue regeneration. Because MSC-CM contain cytokines monocyte chemoattractant protein (MCP)-1, insulin growth factor (IGF)-1, and vascular endothelial growth factor (VEGF) at relatively higher concentrations than other factors, these cytokines were considered as relevant active factors for tissue regeneration. By mixing the recombinant proteins of MCP-1, IGF-1, and VEGF, included at the same concentrations in MSC-CM, we prepared cytokine mixtures mimicking MSC-CM and then evaluated its therapeutic effects in a rat MRONJ model. In vitro, cytokine mixtures promoted osteogenic differentiation, migration, and proliferation of rat MSCs. In addition, these maintained osteoclastic function. In vivo, we used a rat MRONJ model to examine therapeutic effects of the cytokine mixtures through intravenous administration. In MSC-CM or cytokine mixture group, open alveolar sockets in 66% or 67% of the rats with MRONJ, respectively, healed with complete soft tissue coverage and socket bones, whereas in the other groups, the exposed necrotic bone with inflamed soft tissue remained. Histological analysis revealed new bone formation and the appearance of osteoclasts in MSC-CM or cytokine mixture group; however, osteoclasts were significantly reduced in the other groups. Thus, we concluded that intravenous administration of cytokine mixtures might be an effective therapeutic modality for treating patients with MRONJ.

DOI： 10.1002/jbm4.10013

Language：English   Publishing type：Research paper (scientific journal)  

Long-term methotrexate (MTX) treatment can cause MTX-related lymphoproliferative disorder (MTX-LPD). We experienced a case of MTX-LPD that was associated with severe osteonecrosis of the jaw mimicking medication-related osteonecrosis of the jaw. The patient was an 81-year-old woman with rheumatoid arthritis (RA) who was treated with MTX and bisphosphonate. After 7 years, she was referred to our department for the assessment of giant ulcer and exposure of the alveolar bone of the left maxilla. Histopathological and immunological analyses confirmed a diagnosis of MTX-LPD. At seven months after the cessation of MTX treatment, the ulcerative and necrotic lesions had markedly decreased in size. A 1-year follow-up examination showed no evidence of recurrence and good RA control.

DOI： 10.2169/internalmedicine.8946-17

Language：English  

Objectives: The receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitors are novel clinically effective agents that inhibit osteoclast differentiation, function, and survival by binding to RANKL. Medication-related osteonecrosis of the jaw (MRONJ), caused as a result of treatment using denosumab, is a newly emerging type of bone necrosis, the exact pathogenesis of which is unknown. Several studies recently showed that the intravenous administration of mesenchymal stem cells (MSCs) improved the osteonecrosis of the jaw, and it was hypothesized that paracrine effects by secretomes from MSCs are the main constituent. Our aim was to investigate the effects of serum-free conditioned media from human MSCs (MSC-CM) and RANKL inhibitors on osteoclast differentiation. Materials and methods: Cytokines included in MSC-CM were identified using the cytokine array analysis. MSC-CM was added to the culture medium of rat osteoclast precursors containing RANKL inhibitor. Osteoclast differentiation assays, immunohistochemistry, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, and pit formation assays were performed. Results: MSC-CM included various cytokines such as the recruitment of cell osteogenesis angiogenesis and cell proliferation. MSC-CM promoted osteoclast differentiation and expression of master regulatory transcriptional factors for osteoclastogenesis. In addition, MSC-CM showed function maintenance in osteoclasts despite the presence of RANKL inhibitors. Conclusions: Our findings suggest that secretomes in MSC-CM were related to the regulation of osteoclast differentiation, which may reduce the effect of RANKL inhibitors. Clinical relevance: New combinations of drugs using factors included in MSC-CM have effective therapeutic modality for treating patients with MRONJ.

DOI： 10.1007/s00784-016-1986-x

Language：English   Publishing type：Research paper (scientific journal)  

Peripheral nerve regeneration across nerve gaps is often suboptimal, with poor functional recovery. Stem cell transplantation-based regenerative therapy is a promising approach for axon regeneration and functional recovery of peripheral nerve injury; however, the mechanisms remain controversial and unclear. Recent studies suggest that transplanted stem cells promote tissue regeneration through a paracrine mechanism. We investigated the effects of conditioned media derived from stem cells from human exfoliated deciduous teeth (SHED-CM) on peripheral nerve regeneration. In vitro, SHED-CM-treated Schwann cells exhibited significantly increased proliferation, migration, and the expression of neuron-, extracellular matrix (ECM)-, and angiogenesis-related genes. SHED-CM stimulated neuritogenesis of dorsal root ganglia and increased cell viability. Similarly, SHED-CM enhanced tube formation in an angiogenesis assay. In vivo, a 10-mm rat sciatic nerve gap model was bridged by silicon conduits containing SHED-CM or serum-free Dulbecco's modified Eagle's medium. Light and electron microscopy confirmed that the number of myelinated axons and axon-to-fiber ratio (G-ratio) were significantly higher in the SHED-CM group at 12 weeks after nerve transection surgery. The sciatic functional index (SFI) and gastrocnemius (target muscle) wet weight ratio demonstrated functional recovery. Increased compound muscle action potentials and increased SFI in the SHED-CM group suggested sciatic nerve reinnervation of the target muscle and improved functional recovery. We also observed reduced muscle atrophy in the SHED-CM group. Thus, SHEDs may secrete various trophic factors that enhance peripheral nerve regeneration through multiple mechanisms. SHED-CM may therefore provide a novel therapy that creates a more desirable extracellular microenvironment for peripheral nerve regeneration.

DOI： 10.1089/scd.2015.0104

Language：English  

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is defined as an exposed necrotic bone in the oral cavity that does not heal after appropriate intervention for >. 8. weeks with present or previous bisphosphonate treatment in the absence of radiotherapy. Until now, although several risk factors, including invasive dental procedures, infection, mechanical trauma to the jawbone, and concomitant use of immunosuppressive and chemotherapy drugs have been implicated in the etiology of BRONJ, its underlying mechanisms and treatments remain largely unknown. A study recently showed that intravenous administration of mesenchymal stem cells (MSCs) improved BRONJ, and it was hypothesized that paracrine effects by secretomes from MSCs are the main constituent. Here we used rat BRONJ models to examine the therapeutic effects with serum-free conditioned media from human MSCs (MSC-CM), including various secretomes. We showed that MSC-CM has protected rat MSCs and rat osteoclasts. MSC-CM enhanced the expression of osteogenic-related genes and neovascularization-related genes by real-time reverse-transcriptase polymerase chain reaction analysis in in vitro study. In in vivo study, 5-week-old Wistar/ST male rats received zoledronate (35. μg/kg/week) and dexamethasone (1. mg/kg/day) subcutaneously for 2. weeks. Unilateral maxillary molars were then extracted. Two weeks later, rats were divided into non-treatment, serum-free Dulbecco's modified Eagle's medium, and MSC-CM groups. In the MSC-CM group, the open alveolar sockets in 63% of the rats with BRONJ healed with complete soft tissue coverage and socket bones, whereas the exposed necrotic bone with inflamed soft tissue remained in the other groups. Histological analysis showed new bone formation and the appearance of osteoclasts in the MSC-CM group. Osteoclasts were significantly reduced in the non-treatment group. Thus, we concluded that the antiapoptotic and antiinflammatory effects of MSC-CM dramatically regulated the turnover of local bone and indicated therapeutic effects on BRONJ.

DOI： 10.1016/j.bone.2015.01.011

Event date： 2020.11

Language：Japanese  

Venue：WEB開催   Country：Japan  

Event date： 2020.10

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Venue：WEB開催   Country：Japan  

Event date： 2020.4

Language：Japanese  

Country：Japan  

【目的】
シェーグレン症候群（SS）は外分泌腺を特異的に障害される自己免疫疾患であり、自己反応性のT細胞が病体形成の主体とされているが、根治的な治療法は確立されていない。その大きな要因としては、多様なT細胞サブセットから産生される多種のサイトカインが病態形成に関与し、単一の分子を阻害しても疾患を制御できないことが推察される。最近の基礎研究では、幹細胞の培養上清（CM）中の液性因子に免疫抑制作用を有することが報告されている。そこで本研究では、SSの新たな治療法の開発を目的として、骨髄や歯髄由来のCMを用いてT細胞に対する免疫抑制効果を検討した。
【材料と方法】
まず、ヒト骨髄由来間葉系幹細胞培養上清（BMMSC-CM）およびヒト歯髄幹細胞培養上清（DPSC-CM）中の液性分子を網羅的に解析するために、サイトカインアッセイを行った。次に、ヒト末梢血単球細胞をT細胞増殖因子であるphytohemagglutinin (PHA)で刺激培養後、DPSC-CM、BMMSC-CMおよび無血清DMEM存在下でそれぞれ培養し、フローサイトメトリーにてCD3＋CD25＋CD69＋ 活性化 T 細胞の割合を算出した。
【結果】
サイトカインアッセイの結果から、DPSC-CMはBMMSC-CMと比較して、抗炎症作用（IL-10 は34倍、IL-13 は63倍）や細胞増殖能（Hepatocyte growth factorはDPSC-CM のみ、Follistatinは16倍）を有するサイトカイン濃度が高かった。また、フローサイトの結果から、幼若活性化T細胞であるCD3＋CD25＋ では、DPSC-CM は、 BMMSC-CMおよび無血清 DMEM と比較すると、活性化T細胞の割合を減少させることが分かった。また、成熟活性化T細胞であるCD3＋CD69＋でも、DPSC-CM 、BMMSC-CM および無血清 DMEM を比較においても同様の結果が得られた。
【考察】
以上の結果より、DPSC-CMはBMSC-CMより免疫抑制および細胞増殖を有する液性因子が多く含まれ、T細胞の活性化を抑えることが明らかになった。今後はSSモデルマウスにDPSC-CMを腹腔内投与し、唾液腺の病理組織像や唾液量の変化などを評価して、DPSC-CMによる新規治療法の開発を目指す予定である。

Event date： 2020.4

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Country：Japan  

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Country：Korea, Republic of  

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