The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known
as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic
stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional
differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman
spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs
were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a
Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM
Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined
intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The
differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold
embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands
which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine,
guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified.
The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process.
Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with
different degrees of maturation.
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