In situ spectral monitoring of mRNA translation in embryonic stem cells during differentiation in vitro.

Abstract

Raman microspectroscopy was used to determine biochemical markers during the differentiation of embryonic murine stem cells (mES) in vitro. Such markers are useful to determine the differentiation status of ES cells cultured on biomaterials. Raman spectra of mES cells as undifferentiated, spontaneously differentiated (4 days), and differentiated cells via formation of embryoid bodies (16, 20 days) were analyzed. Unsupervised hierarchical cluster analysis and principal component analysis were used to determine biochemical differences between mES cells in various states of differentiation. The undifferentiated cells were characterized by high scores of the first principal component (PC1, 49% variance). Similarity between the PC1 loading and the Raman spectrum of RNA indicated a high concentration of RNA in mES cells compared to differentiated cells. The ratio between the peak areas of RNA and proteins was used as a measure of mRNA translation. Using the same peak area ratio, it was possible to differentiate even between mES as undifferentiated and in early stages of differentiation (4 days). These findings were correlated with biological studies reporting high levels of nontranslated mRNA during early embryonic development. Therefore, the RNA translation obtained from the Raman spectra can be used as marker of differentiation state of mES cells.