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

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.

Keywords: Autophagy; Mouse embryonic palatal mesenchyme cells; PTEN-Akt-mTOR signaling pathway; Stemness.

Fig. 1

Identity and purity of MEPM cells. a MEPM cells emerging from palatal shelves. b CCK-8 assay profile showing MEPM cells proliferation at passage 1. Scale bar: 100 μm

Fig. 2

The properties of passage 1 MEPM cells. a Immunofluorescence staining of MEPM cells for vimentin (green), nestin (red), HNK-1 (green) (white arrow), and keratin (not stained). b Immunofluorescence staining of MEPM cells for PDGFRα (green), Efnb1 (red), Osr2 (red), and Meox2 (red). c Transwell assays showing that PDGFA (0.5 ng/mL and 5 ng/mL) stimulates chemotaxis of MEPMs. N = 9; *P < 0.05; **P < 0.01. Cell nuclei (blue) were counterstained with DAPI, and HNK-1 (red) in cytoplasm was stained with Evans blue dye. Scale bar: 20 μm

Fig. 3

Immunophenotyping of MEPM cells. a Raw flow cytometry profiles of MEPM cells. b Semi-quantitative profiles from raw flow cytometry data showing cell surface marker expression levels in MEPM cells. Isotype gating using IgG-FITC and IgG-PE controls was performed to minimize non-specific signal

Fig. 4

Adipogenic differentiation of MEPM cells. a Oil Red O-positively stained MEPM cells cultured in adipogenic-inducing (left panel) medium for 3 weeks and Oil Red O-negative MEPM cells cultured in regular culture medium (right panel). Scale bar: 50 μm. b qRT-PCR profile showing the expression levels of adiponectin and LPL in the induced and non-induced groups; N = 3; *P < 0.05; **P < 0.01

Fig. 5

Osteogenic differentiation of MEPM cells. a ALP staining of induced (left panel) and non-induced (right panel) MEPM cells after 2 weeks of osteogenic induction; scale bar: 50 μm. b AR staining of induced (left panel) and non-induced (right panel) MEPM cells after 3 weeks of osteogenic induction (scale bar: 50 μm). c qRT-PCR profile showing ALP and Cbfα-1 expression levels in the induced and non-induced groups; N = 3; **P < 0.01

Fig. 6

Chondrogenic differentiation of MEPM cells. a Immunohistochemical staining of Col-II in induced (left panel) and non-induced (right panel) groups; scale bar: 50 μm. b qRT-PCR profile showing COMP and Col-II expression levels in the induced and non-induced groups; N = 3; *P < 0.05; **P < 0.01

Fig. 7

Characteristics of autophagy in MEPM cells. a LC3-II immunofluorescence staining before differentiation and after osteogenic and adipogenic differentiation. Cell nuclei (blue) were counterstained with DAPI; cytoplasm (red) was stained with Evans blue; scale bar: 20 μm. b Western blot showing expression levels of LC3-I/II and p62 proteins before and after osteogenic differentiation; N = 3; *P < 0.05; **P < 0.01. c TEM image showing large amount of autophagosomes (red arrows) before osteogenic differentiation, which decreased sharply after osteogenic differentiation

Fig. 8

Characteristics of MEPM cells at passage 4. a LC3-II immunofluorescence staining; scale bar: 20 μm. b AR staining of induced, non-induced, and VO-OHpic trihydrate-treated MEPM cells after 3 weeks of osteogenic induction; scale bar: 50 μm. c qRT-PCR profile showing ALP and Cbfα-1 expression levels in the induced, non-induced, and VO-OHpic trihydrate-treated groups; N = 3; **P < 0.01

Fig. 9

PTEN-Akt-mTOR autophagic signaling. a Western blot showing the levels of phosphorylated and non-phosphorylated PTEN, AKT, and mTOR proteins before and after osteogenic differentiation. b Western blot showing the levels of phosphorylated and non-phosphorylated PTEN, AKT, and mTOR proteins before and after VO-OHpic trihydrate treatment. c Schematic illustration of the autophagic pathway. N = 3; *P < 0.05; **P < 0.01

Fig. 10

RA-induced cleft palate in Kunming mice. a Comparison of general palatal morphology between the normal group and RA-induced group. b Goldner’s trichrome staining of frontal sections of palatal regions at E15.5 and E17.5 in normal palate and RA-induced cleft palate. Mineralized bones are stained green; osteoid are stained orange/red; chondrocyte are stained purple; nuclei are stained blue/gray; and the cytoplasm is stained red/pink. Scale bar: 200 μm