Bioactive calcium silicate extracts regulate the morphology and stemness of human embryonic stem cells at the initial stage

Embryonic stem cells (ESCs) are undifferentiated cells that have the capacity to self-renew and differentiate into a variety of cells and provide cell sources for regenerative medicine or biological specimens for cytotoxicity tests. Calcium silicate (CS), a bioactive silicate ceramic, can stimulate the osteogenic differentiation of various types of stem cells, but its role in regulating the biological phenotypes of ESCs remains unclear. Here, the impact of CS on human ESCs was investigated using CS-supplemented medium. The cytotoxicity of CS to hESCs and its effects on apoptosis, growth, proliferation, and differentiation were quantified systematically. Morphological analysis of hESC colonies indicated that the bioactive ions released from CS have little cytotoxicity to hESCs at two CS concentrations. Immunofluorescence and flow cytometry analyses showed that apoptosis was time-independent at early or late stages of hESC growth. In contrast, CS ion extracts regulated hESC differentiation in a time-dependent manner: ESC stemness was preserved by enhancing Oct-4, Sox-2, and Nanog gene expression at day 3, while the cells tended to differentiate at day 6. Combined tests on gene and protein levels further indicated that hESCs tended to differentiate into mesoderm in the presence of CS ion extracts, especially at low CS concentrations. These results demonstrate the effects of CS extracts on hESC stemness and differentiation at the molecular and cellular levels, suggesting that CS-based biomaterials could serve as a potential regulator for ESCs in regenerative medicine.