Cytochemical and ultrastructural characterization of growing colonies of human embryonic stem cells

Abstract

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.

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

“…Single or clustered rounded protrusions in the cell membrane, in particular the 14-day-old cultures ( Fig. 7D), showed similarity to the coarse particles and halos previously identified by phase contrast microscopy consisting of apoptotic cells and bodies (Johkura et al, 2004). This suggests a pronounced cell death at this late stage.…”

“…However, according to our TEM investigations, these peripheral monolayers of the intermediate stage colonies grown on MEF exhibited junctional complexes, hence suggesting an epithelial nature. Colonies organized with confined undifferentiated cells and peripheral differentiation was confirmed by a shift in immunoreactivity from the pluripotency marker SSEA-4 to a marker of differentiation SSEA-1 towards the periphery (Johkura et al, 2004). This downregulation in pluripotency markers was to some extent also indicated in the short-term feeder free cultures referred to above, where the intensity in immunostainings of the key pluripotency markers OCT4 and NANOG decreased in peripheral regions (Ullmann et al, 2007).…”

“…Despite an apparent decrease in the amount of the key transcription factors, expression levels were still substantial, raising the question whether a deviation from the undifferentiated morphology to a more epithelial-like morphology is accompanied by decrease in potency. Additionally, increased amounts of laminin in the periphery of the colonies confirmed accumulation of extracellular matrices under standard culture conditions (Johkura et al, 2004), most likely as a constituent of the basement membrane secreted by differentiated epithelial cells. One of the intermediate (11 days in culture) and the late stage colonies had distinct basement membranes.…”

Hydroxyapatite nanoparticles inhibit the growth of human glioma cells in vitro and in vivo

“…When U251 and SHG44 cells were anchored to plates, the different concentrations (0, 120, and 240 mg/L) of nano-HAPs were added, and the U251 and SHG44 cells were incubated for 48 hours; the specimens were postfixed in 1% osmium tetroxide/0.1 M sodium cacodylate buffer, dehydrated in a graded series of ethanol, and embedded after rinsing three times. 10 The ultrathin sections of the specimens were stained with lead citrate and uranyl acetate, and observed with a transmission electron microscope. Each assay was performed in triplicate.…”

Regulation of Apoptosis and Differentiation by p53 in Human Embryonic Stem Cells

“…Previously, basic fibroblast growth factor and activin A were identified as self-renewal factors (3)(4)(5)(6). However, for reasons that are not clear, hESCs often display high rates of spontaneous apoptosis and differentiation in culture, thus making the process of expanding these cells highly inefficient (3,(7)(8)(9)(10). For example, Dravid et al (8) reported that, under routine culture conditions, Ͼ30% of hESCs undergo spontaneous apoptosis.…”