From Pluripotency to Early Differentiation of Human Embryonic Stem Cell Cultures Evaluated by Electron Microscopy and Immunohistochemistry

Under appropriate culture conditions human embryonic stem cells (hESCs) can retain an undifferentiated state during numerous passages (Thomson et al., 1998). In the undifferentiated state, hESCs express characteristic markers like NANOG, OCT4, TDGF1, DNMT3B, GABRB3, and GDF3, and are maintained by plating undifferentiated cells or colonies of cells into new culture dishes with fresh medium every 7 to 10th day (Adewumi et al., 2007). Following periods exceeding 7 to 10 days in culture without passage, the cell population tends to become heterogeneous with differentiation starting to occur within a given colony or in various parts of a culture dish. The tendency for undergoing differentiation is independent of whether feeder cells, protein matrixes, or special plastic surfaces are used and what specific hESC medium is employed. Although an increasing density of cells during culture has been suggested to be one reason for spontaneous differentiation of cells to occur, it is also well known that morphologically perfect undifferentiated hESCs often appear in very high density in the same culture dish, even when differentiation has started to occur (Laursen et al., 2007). The transition from the undifferentiated state to more differentiated cell types appears to take place as a gradual process in colonies of hESCs, and it is currently not known how the ultrastructural organization of cells changes along with the differentiation process as defined from immunohistochemical differentiation markers. In the present study we have performed a spatiotemporal investigation on the differentiation of hESC colonies by electron microscopical and immunohistochemical approaches.