Abstract
BackgroundBoth genetic and environmental factors are implicated in the pathogenesis of cleft palate. However, the molecular and cellular mechanisms that regulate the development of palatal shelves, which are composed of mesenchymal cells, have not yet been fully elucidated. This study aimed to determine the stemness and multilineage differentiation potential of mouse embryonic palatal mesenchyme (MEPM) cells in palatal shelves and to explore the underlying regulatory mechanism associated with cleft palate formation.MethodsPalatal shelves excised from mice models were cultured in vitro to ascertain whether MEPM are stem cells through immunofluorescence and flow cytometry. The osteogenic, adipogenic, and chondrogenic differentiation potential of MEPM cells were also determined to characterize MEPM stemness. In addition, the role of the PTEN-Akt-mTOR autophagic pathway was investigated using quantitative RT-PCR, Western blotting, and transmission electron microscopy.ResultsMEPM cells in culture exhibited cell surface marker expression profiles similar to that of mouse bone marrow stem cells and exhibited positive staining for vimentin (mesodermal marker), nestin (ectodermal marker), PDGFRα, Efnb1, Osr2, and Meox2 (MEPM cells markers). In addition, exposure to PDGFA stimulated chemotaxis of MEPM cells. MEPM cells exhibited stronger potential for osteogenic differentiation as compared to that for adipogenic and chondrogenic differentiation. Undifferentiated MEPM cells displayed a high concentration of autophagosomes, which disappeared after differentiation (at passage four), indicating the involvement of PTEN-Akt-mTOR signaling.ConclusionsOur findings suggest that MEPM cells are ectomesenchymal stem cells with a strong osteogenic differentiation potential and that maintenance of their stemness via PTEN/AKT/mTOR autophagic signaling prevents cleft palate development.
Highlights
Both genetic and environmental factors are implicated in the pathogenesis of cleft palate
No keratin-stained cells were observed after passage 1; similar proportions of vimentin, nestin, and HNK-1-positive cells were observed after passage 1 (Fig. 2a), which suggests that cell passaging enabled mouse embryonic palatal mesenchyme (MEPM) cell specialization
Transwell assay revealed that exposure to PDGFA stimulated chemotaxis of MEPM cell chemotaxis; this confirmed our immunofluorescence results that showed positive expression of PDGFRα on the isolated MEPM cells, which is consistent with the findings of a previous study [27] (Fig. 2c)
Summary
Both genetic and environmental factors are implicated in the pathogenesis of cleft palate. This study aimed to determine the stemness and multilineage differentiation potential of mouse embryonic palatal mesenchyme (MEPM) cells in palatal shelves and to explore the underlying regulatory mechanism associated with cleft palate formation. Other studies have found no association between cell death and palatal mesenchyme development except in the vicinity of the medial epithelial seam (MES) [16, 17]. These studies did not involve in-depth characterization of the stemness and other characteristics of MEPM cells that may potentially influence cleft palate formation. We investigated the multilineage differentiation potential of MEPM cells to determine their stemness and examined the underlying mechanisms that regulate palate development
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