9 research outputs found

    Remission for Loss of Odontogenic Potential in a New Micromilieu <i>In Vitro</i>

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    <div><p>During embryonic organogenesis, the odontogenic potential resides in dental mesenchyme from the bud stage until birth. Mouse dental mesenchymal cells (mDMCs) isolated from the inductive dental mesenchyme of developing molars are frequently used in the context of tooth development and regeneration. We wondered if and how the odontogenic potential could be retained when mDMCs were cultured <i>in vitro</i>. In the present study, we undertook to test the odontogenic potential of cultured mDMCs and attempted to maintain the potential during culturing. We found that cultured mDMCs could retain the odontogenic potential for 24 h with a ratio of 60% for tooth formation, but mDMCs were incapable of supporting tooth formation after more than 24 h in culture. This loss of odontogenic potential was accompanied by widespread transcriptomic alteration and, specifically, the downregulation of some dental mesenchyme-specific genes, such as <i>Pax9</i>, <i>Msx1</i>, and <i>Pdgfrα</i>. To prolong the odontogenic potential of mDMCs <i>in vitro</i>, we then cultured mDMCs in a serum-free medium with Knockout Serum Replacement (KSR) and growth factors (fibroblastic growth factor 2 and epidermal growth factor). In this new micromilieu, mDMCs could maintain the odontogenic potential for 48 h with tooth formation ratio of 50%. Moreover, mDMCs cultured in KSR-supplemented medium gave rise to tooth-like structures when recombined with non-dental second-arch epithelium. Among the supplements, KSR is essential for the survival and adhesion of mDMCs, and both Egf and Fgf2 induced the expression of certain dental mesenchyme-related genes. Taken together, our results demonstrated that the transcriptomic changes responded to the alteration of odontogenic potential in cultured mDMCs and a new micromilieu partly retained this potential <i>in vitro</i>, providing insight into the long-term maintenance of odontogenic potential in mDMCs.</p></div

    KSR, Fgf2 and Egf facilitate the maintenance of odontogenic potential in cultured mDMCs.

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    <p><b>(A)</b> The relational network was constructed based on the strength of correlation between pairs of samples, showing the relationship of mDMCs to normal neural crest cell types. <b>(B)</b> mDMCs proliferated at a significantly higher rate in KSR-supplemented medium (M-KSR) than FBS-supplemented medium (M-FBS). <b>(C)</b> The mRNA levels of some dental mesenchyme-related genes in mDMCs cultured in medium with KSR. <b>(D)</b> Immunofluorescence analysis of Msx1, Pax9, and Pdgfrα in mDMCs cultured in medium with KSR or FBS. <b>(E)</b> Recombinants with mDMCs cultured for 24 h and 48h in KSR-supplemented medium developed into teeth after subrenal culture for 3 weeks. R, ratio of tooth formation. <b>(F)</b> Recombinants of epithelium from E10.5 mouse second-arch (mse) and cultured mDMCs developed into tooth-like structures. *<i>p</i> < 0.05 compared with the freshly isolated cells. Scale bar: D = 100 μm; E, F = 500 μm.</p

    Design of the study.

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    <p>Dental mesenchyme tissues from embryonic day 14.5 (E14.5) mice were digested with trypsin. Freshly isolated mouse dental mesenchymal cells (mDMCs) were divided into three groups: One group was recombined with embryonic dental epithelial and cultured in kidney; the second was submitted for RNA-seq; the third was cultured <i>in vitro</i> and harvested at indicated time points.</p

    Impaired odontogenic potential in cultured mDMCs.

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    <p><b>(A)</b> mDMCs possess a fibroblast-like or elliptic morphology. Cell density increased significantly and the morphological alteration was visible from 24 h to 96 h. <b>(B)</b> Cells continued to proliferate in culture and cell quantity doubled in 48 h. <b>(C)</b> Recombinants with freshly isolated mDMCs and cells cultured for 24 h formed tooth after subrenal culture, but recombinants with cells cultured for 48 h formed cysts. Recombinants with E14.5 dental mesenchyme (Tis) were used as positive control. H&E staining showed the presence of tooth structure or eosinophilic amorphous matrix. D, dentin; PD, predentin; DP, dental pulp; OD, odontoblast. Scale bar: A, B = 50 μm; C = 500 μm.</p

    Phenotypic alteration of cultured mDMCs.

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    <p><b>(A)</b> Heat map showed the decreased expression of dental mesenchyme-related genes in cultured mDMCs. <b>(B)</b> The mRNA levels of some dental mesenchyme-related genes. <b>(C)</b> Decreased expression of Msx1, Pax9, P75, and Pdgfrα in cultured mDMCs was examined by immunofluorescence assays. *<i>p</i> < 0.05 compared with the D0 cells. Scale bar: 100 μm.</p

    The effects of KSR, Fgf2, and Egf on cultured mDMCs.

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    <p><b>(A)</b> Images of cells cultured in the medium with Egf/Fgf2, KSR/Egf, KSR/Fgf2, and KSR/Egf/Fgf2. Scale bar: 50 μm. (B) The mRNA levels of some dental mesenchyme-related genes in freshly isolated (D0) mDMCs and mDMCs cultured in KSR-, KSR/Fgf2-, KSR/Egf-, and KSR/Egf/Fgf2-supplemented medium. *<i>p</i> < 0.05 compared with the D0 cells.</p

    Global transcriptome profiles of mDMCs.

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    <p><b>(A)</b> Hierarchical clustering analysis of dental mesenchymal tissue or cell samples. Tis, dental mesenchymal tissues. <b>(B)</b> Principal components analysis (PCA) showed the distances between various cell populations. Variation between two adjacent cell populations was reduced with time. <b>(C)</b> The number of differentially expressed genes between every two cell populations was listed. The red bracket indicates the number of upregulated genes and the blue indicates the downregulated genes. <b>(D)</b> Differentially expressed genes were divided into eight clusters according to their temporal patterns. Functional annotation analysis of genes in each cluster was performed, and the biological processes were reconstructed. The red bracket indicates processes involving the upregulated genes in the given time interval and the blue bracket indicates those involving the downregulated genes. * indicated the biological processes associated with tooth development.</p
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