Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy |
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Authors: | Flavius C Pascut Spandan Kalra Vinoj George Nathan Welch Chris Denning Ioan Notingher |
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Institution: | 1. School of Physics and Astronomy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK;2. Wolfson Centre for Stem Cells, Tissue Engineering & Modelling, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, NG7 2RD, UK |
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Abstract: | BackgroundOnline label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.MethodsEBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5 days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24 h intervals.ResultsThe Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340 cm− 1, 1083 cm− 1, 937 cm− 1, 858 cm− 1, 577 cm− 1 and 482 cm− 1. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.ConclusionsThe intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.General significanceAs the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems. |
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Keywords: | Embryoid body Human embryonic stem cell Differentiation Cardiomyocyte Raman spectroscopy |
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