Blackwell Publishing, Ltd. Cytochemical and ultrastructural characterization of growing colonies of human embryonic stem cells

The morphology of human embryonic stem (ES) cells changes with their colonial growth. For a better understanding of the growth of ES cell colonies in culture, we determined their cytochemical and ultrastructural characteristics focusing on images of living cells under a phase contrast microscope. During the initial growth stages, the colonies exhibited a mosaic appearance with discernible cell-cell borders. PAS staining coupled with amylase digestion demonstrated that the bright granules and dark deposits in the cytoplasm contained glycogen. Ultrastructurally they were glycogen accumulations, and clustered open spaces associated with various amounts of glycogen. Although intercellularly heterogeneous, these structures were detectable throughout colony growth. As the colonies grew, compaction towards the centre emerged and increased, accompanied by heterogeneous increases in coarse particles with or without a halo. TUNEL showed these particles to consist at least in part of apoptotic cells/bodies. Transmission electron microscopy indicated that most apoptotic cells had been phagocytosed by intact ES cells. Spontaneous differentiation was detected occasionally in the periphery of the colonies. The presence of PASpositive fibrous structures not susceptible to amylase digestion and laminin-immunoreactivity indicated the accumulation of extracellular matrix in the peripheral differentiated areas. These findings made it possible to determine the growth stage of human ES cell colonies.