Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy
| Autor: | Flavius C. Pascut, Chris Denning, Spandan Kalra, Nathan Welch, Vinoj George, Ioan Notingher |
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| Rok vydání: | 2013 |
| Předmět: |
Cell Culture Techniques
Biophysics Analytical chemistry Cell Differentiation Embryoid body Biology Spectrum Analysis Raman Antigens Differentiation Biochemistry Embryonic stem cell In vitro Cell Line Cell biology symbols.namesake Chemically defined medium symbols Humans Actinin Myocytes Cardiac Raman microscope Stem cell Raman spectroscopy Molecular Biology Embryonic Stem Cells Label free |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - General Subjects. 1830:3517-3524 |
| ISSN: | 0304-4165 |
| Popis: | 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 5 days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24 h 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 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. 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. |
| Databáze: | OpenAIRE |
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