Human embryonic stem cells (hESCs) have large nucleus-to-cytoplasm ratios and nucleic acid spectral bands are prominent in their characteristic Raman signatures. Under normal conditions, the major variations in these signatures are due to changes in glycogen content, but how these signatures vary in response to different external conditions is largely unknown. In this study we investigated the influences of temperature variations on hESC Raman signatures. At 32 °C, compared to the 37 °C control condition, cell proliferation rates were markedly reduced and glycogen Raman band intensities were elevated. In addition, at both temperatures, an inverse relationship between cell proliferation rates (i.e., onset of exponential growth phase vs. end of exponential phase) and glycogen Raman band intensities was observed. This relationship suggested a role for glycogen in the energy metabolism of hESC self-renewal. Protein and lipid spectral variations were small and co-varied with those of nucleic acids, suggesting that they were related to changes in cellular dimensions occurring during the cell cycle. When the temperature was elevated to 39 °C, increased glycogen band intensities, compared to controls, were also observed. In addition, spectral evidence of differentiation emerged that was supported by reduced SSEA-3 expression. Taken together, these results demonstrated that heat and cold stress had quite different effects on the characteristic Raman signatures of hESCs. Thus, Raman spectroscopy can be used to detect deviation from optimal culturing temperatures and therefore it could be of considerable value in the routine and noninvasive determination of hESC culture quality.

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