Osteogenic microenvironment affects palatal development through glycolysis

Palate development involves various events, including proliferation, osteogenic differentiation, and epithelial-mesenchymal transition. Disruption of these processes can result in the cleft palate (CP). Mouse embryonic palatal mesenchyme (MEPM) cells are commonly used to explore the mechanism of palatal development and CP. However, the role of the microenvironment in the biological properties of MEPM cells, which undergoes dynamic changes during palate development, is rarely reported. In this study, we investigated whether there were differences between the palatal shelf mesenchyme at different developmental stages. Our results found that the palatal shelves facilitate proliferation at the early palate stage at mouse embryonic day (E) 13.5 and the tendency towards osteogenesis at E15.5, the late palate development stage. And the osteogenic microenvironment, which was mimicked by osteogenic differentiation medium (OIM), affected the biological properties of MEPM cells when compared to the routine medium. Specifically, MEPM cells showed slower proliferation, shorter S phase, increased apoptosis, and less migration distance after osteogenesis. E15.5 MEPM cells were more sensitive than E13.5, showing an earlier change. Moreover, E13.5 MEPM cells had weaker osteogenic ability than E15.5, and both MEPM cells exhibited different Lactate dehydrogenase A (LDHA) and Cytochrome c (CytC) expressions in OIM compared to routine medium, suggesting that glycolysis might be associated with the influence of the osteogenic microenvironment on MEPM cells. By comparing the stemness of the two cells, we investigated that the stemness of E13.5 MEPM cells was stronger than that of E15.5 MEPM cells, and E15.5 MEPM cells were more like differentiated cells than stem cells, as their capacity to differentiate into multiple cell fates was reduced. E13.5 MEPM cells might be the precursor cells of E15.5 MEPM cells. Our results enriched the understanding of the effect of the microenvironment on the biological properties of E13.5 and E15.5 MEPM cells, which should be considered when using MEPM cells as a model for palatal studies in the future.