Background: Online 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.
Methods: EBs 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 5days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24h intervals.
Results: The 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 1340cm(-1), 1083cm(-1), 937cm(-1), 858cm(-1), 577cm(-1) and 482cm(-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.
Conclusions: The 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 significance: As 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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