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Objective: Cancer‐associated fibroblasts (CAFs) are abundantly infiltrated in oral squamous cell carcinoma (OSCC), but the contact‐dependent mechanisms that regulate CAFs phenotype in precursor cells, such as paracancerous fibroblasts (PFs), remain unclear. Here, a fibroblast‐attached organoid (FAO) model was initiated to determine phenotype transition of fibroblasts triggered by contact with OSCC. Material and methods: Organoids and fibroblasts were generated using OSCC and adjacent tissues. Cell‐clusters containing fibroblasts and tumour cells were aggregated to allow for FAOs expansion. Immunoblotting assay was performed to compare expression of Notch intracellular domain (NICD) in CAFs and PFs. Colony formation assay was employed to evaluate morphological activation of fibroblasts. Results: Compared to traditional 3D co‐culture, FAOs better modulated the spatial distribution of fibroblasts with tumour nests. The presence of CAFs with multiple branches was stably observed in FAOs during serial passage. Incorporation with organoids promoted the ability of PFs to form multiple branches. Immunoblotting assay confirmed higher NICD level in CAFs than PFs. Treatment with Notch inhibitor, N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (i.e. DAPT) blocked morphological activation of fibroblasts incorporated into FAO. Conclusion: We developed a robust strategy to study contact‐dependent mechanisms underlying tumour‐stromal interaction, and suggested that Notch activity contributes to biogenesis of OSCC‐associated fibroblasts. [ABSTRACT FROM AUTHOR]

Objectives We examined whether mouse embryonic stem ( ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Materials and methods Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Results Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Conclusions Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. [ABSTRACT FROM AUTHOR]