In situ spectroscopic study of nucleic acids in differentiating embryonic stem cells

The in vitro differentiation of embryonic stem (ES) cells into specific phenotypes plays an important role in the development of stem cell therapy, tissue engineering and regenerative medicine. Currently, there are no biological assays able to characterise and monitor in situ and in real-time ES cells during the differentiation process. We applied Raman micro-spectroscopy to compare undifferentiated murine ES cells with murine ES cells in the differentiation process. The most significant differences between undifferentiated and differentiated ES cells (16 days differentiation via formation of embryoid body) were related to the nucleic acids. The decrease in the magnitude of RNA 813 cm(-1) Raman peaks (25%) in the differentiated ES cells in comparison to undifferentiated ES cells, suggests that part of the RNA in the ES cells is used for the synthesis of specific proteins in the early stages of differentiation. In the same time, the DNA 786 cm(-1) Raman peaks were lower by 50%, indicating that the differentiated cells are more in the G1 phase than S, G2 or M phases of the cell cycle. This result suggests that the proliferation rate of differentiated cells is reduced following development of a mature phenotype. This study shows the feasibility of using Raman micro-spectroscopy to monitor in situ and in real-time the differentiation of ES cells by using the intensity of Raman peak of nucleic acids as differentiation markers. (C) 2004 Elsevier B.V. All rights reserved.