Raman microspectroscopy is a marker free method

Raman microspectroscopy is a marker-free method that can be used to characterize single cells based on a pattern of molecular vibrational modes, which reflects the composition of intracellular proteins, lipids, nucleic acids, and adenosine triphosphate (Puppels et al., 1990). Notingher et al. (2004) investigated changes in Raman spectra due to cellular differentiation and demonstrated that the method, in combination with principal component analysis (PCA), can be used as a tool for discriminating pluripotent cells from their cardiac progeny (Pascut et al., 2011). Our group has integrated a custom-made Raman spectroscopic system with a fluorescence microscope to demonstrate that Raman signal patterns can be correlated to specific cell phenotypes and stages (Brauchle et al., 2014). Here, we employed Raman microspectroscopy to acquire biochemical fingerprints of the right atrium (RA), right ventricle (RV), left atrium (LA), and left ventricle (LV) of murine and human heart tissues. We further assessed biochemical shifts specific for cardiovascular lineage commitment and cardiac specification in differentiating murine and human ESCs (mESCs and hESCs) employing PCA on the spectral data (Figure S1). The unique combination of Raman spectroscopy with high-resolution fluorescence microscopy allowed the collection of Raman profiles of mESC- and hESC-derived CMs with an atrial or ventricular specification. Raman patterns and spectral differences were verified by analyzing fetal murine (mfCMs) and human CMs (hfCMs). We further identified that alterations of cardiac protein expression patterns, which occur after birth when CMs adapt to their specific physiological tasks (Sylva et al., 2014), also correlate to specific shifts in the cardiac Raman signature and thus Raman microspectroscopy was used to assess the maturity of the in vitro-generated ESC-derived CMs.
Results
Discussion Nevertheless, technological improvements and adaptions will be necessary to achieve the transition of Raman microspectroscopy from a tool for research and development purposes to a routine device for the marker-free single-cell sorting and high-throughput analysis. In a very elegant study, Dochow et al. (2013) showed the feasibility of Raman spectroscopy and cell sorting in a microfluidic chamber. The further reduction of measurement times and the establishment of automated software to facilitate spectral analysis, and for non-experienced users, are currently addressed by other groups (Ren et al., 2014; Richardson et al., 2014). Our microspectroscopic approach shows that mESCs, hESCs, and their cardiac progeny exhibit unique molecular fingerprints, and the acquisition of Raman spectral data from such biological materials provides insights into molecular and developmental cell biology processes, potentially enabling the identification of molecular drivers in cell differentiation pathways (Sule-Suso et al., 2014). The Raman spectra obtained from ESCs were consistent with data published by others (Chan et al., 2009). As previously described, mESC molecular fingerprint patterns showed characteristic signals that can be attributed to nucleic acids (Notingher et al., 2004). Here, we detected only median nucleic acid peaks, but stronger signals from lipids at 1300, 1437, and 1658 cm−1 (Krafft et al., 2005) in hESCs. Culture conditions as well as cell line-specific variations, which are controversially discussed for pluripotent stem cells (Burridge et al., 2012), could impact the molecular Raman signature. Tan et al. (2012) showed that differences in the spectra of hESCs and hiPSCs are only minimal compared with the spectral shifts that occur due to cell differentiation. This is consistent with our data presented in this study, where cardiac differentiation led to specific molecular changes in the cellular fingerprint spectrum of pluripotent cells. Namely, cardiac cell fate commitment was detectable by an increase of the Raman bands at 701, 1065, and 1437 cm−1 in human and murine cells. These Raman bands can be assigned to cholesterol but also other lipid molecules (Krafft et al., 2005). Although it has been described that lipids play a role in energy storage and homeostasis in cardiac muscle, the role of lipid metabolism and energy storage in cardiac cell fate decisions has not been fully understood. The metabolic machinery of ESCs, in contrast to ESC-CMs, is reported to be only rudimentarily developed (Chung et al., 2007). In cardiac differentiation, successive remodeling of this metabolic system has been identified to orchestrate the alignment of mitochondria and cardiac-specific key regulators for high energy-dependent CMs (Chung et al., 2007). Cholesterol-rich membrane domains, cholesterol signaling, and lipid metabolism might play a role in early cardiac cell fate decisions as the Raman profiles of ESCs, CPCs, and ESC-CMs vary significantly. To our knowledge, this is the first report in which Raman microspectroscopy was employed for CPC characterization. To further elucidate CPC subpopulations, the spectroscopic data could be correlated with data of cellular metabolism and protein expression to subspeciate this heterogeneous population.