鼠胚胎腭中胚层间充质细胞通过 PTEN-Akt-mTOR 自噬途径维持干细胞特性。

Mouse embryonic palatal mesenchymal cells maintain stemness through the PTEN-Akt-mTOR autophagic pathway.

机构信息

Department of Plastic Surgery and Burn Center, the Second Affiliated Hospital of Shantou University Medical College, North Dongxia Road, Shantou, 515041, Guangdong, China.

Shantou University Medical College, No. 22 Xinling road, Shantou, 515041, Guangdong, China.

出版信息

Stem Cell Res Ther. 2019 Jul 29;10(1):217. doi: 10.1186/s13287-019-1340-8.

DOI:10.1186/s13287-019-1340-8

PMID:31358051

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6664599/

Abstract

BACKGROUND

Both 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.

METHODS

Palatal 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.

RESULTS

MEPM 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.

CONCLUSIONS

Our 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.

摘要

背景

遗传和环境因素都与腭裂的发病机制有关。然而，调节由间充质细胞组成的腭突发育的分子和细胞机制尚未完全阐明。本研究旨在确定腭突中胚胎鼠腭间质（MEPM）细胞的干性和多能分化潜能，并探讨与腭裂形成相关的潜在调节机制。

方法

从腭裂模型小鼠中取出腭突进行体外培养，通过免疫荧光和流式细胞术确定 MEPM 是否为干细胞。还确定了 MEPM 细胞的成骨、成脂和成软骨分化潜能，以表征 MEPM 干性。此外，还通过定量 RT-PCR、Western blot 和透射电镜研究了 PTEN-Akt-mTOR 自噬通路的作用。

结果

培养中的 MEPM 细胞表现出与小鼠骨髓干细胞相似的细胞表面标志物表达谱，并对波形蛋白（中胚层标志物）、巢蛋白（外胚层标志物）、PDGFRα、Efnb1、Osr2 和 Meox2（MEPM 细胞标志物）呈阳性染色。此外，PDGFA 暴露刺激 MEPM 细胞的趋化性。与成脂和成软骨分化相比，MEPM 细胞表现出更强的成骨分化潜能。未分化的 MEPM 细胞显示出高浓度的自噬体，这些自噬体在分化后（在第四代）消失，表明 PTEN-Akt-mTOR 信号通路的参与。