Non-invasive Chamber-Specific Identification of Cardiomyocytes in Differentiating Pluripotent Stem Cells

Abstract

SummaryOne major obstacle to the application of stem cell-derived cardiomyocytes (CMs) for disease modeling and clinical therapies is the inability to identify the developmental stage of these cells without the need for genetic manipulation or utilization of exogenous markers. In this study, we demonstrate that Raman microspectroscopy can non-invasively identify embryonic stem cell (ESC)-derived chamber-specific CMs and monitor cell maturation. Using this marker-free approach, Raman peaks were identified for atrial and ventricular CMs, ESCs were successfully discriminated from their cardiac derivatives, a distinct phenotypic spectrum for ESC-derived CMs was confirmed, and unique spectral differences between fetal versus adult CMs were detected. The real-time identification and characterization of CMs, their progenitors, and subpopulations by Raman microspectroscopy strongly correlated to the phenotypical features of these cells. Due to its high molecular resolution, Raman microspectroscopy offers distinct analytical characterization for differentiating cardiovascular cell populations.

Steps toward Maturation of Embryonic Stem Cell-Derived Cardiomyocytes by Defined Physical Signals

“…In accordance, d20 dyn hESC-CMs and human fCMs showed stronger structural protein- and lipid-related peak intensities, but decreased signals of nucleotide bands when compared with hESC-CMs cultured for only 10 days or under static conditions. The observed differences in protein-assigned bands are possibly due to the sarcomeric organization and increasing myofibril densities in the d20 dyn hESC-CMs, as was confirmed by IF staining in this study and as hypothesized previously (Brauchle et al., 2016). These findings highlight the importance of combined pulsatile flow and cyclic strain in the maturation process of PSC-CMs.…”

“…The investigation of the correlated PC-1 loading patterns elucidated a stronger impact of peaks at 860 cm −1 (glycogen), 938 cm −1 (glycogen), 1,003 cm −1 (phenylalanine), 1,123 cm −1 (glycogen), and 1,658 cm −1 (amide I) on the spectra of d18 dyn mESC-CMs when compared with those from d18 stat mESC-CMs. These peaks were all previously associated with postnatal maturation of CMs (Figure 2E; Notingher et al., 2004, Movasaghi et al., 2007, Pascut et al., 2013, Brauchle et al., 2016). In contrast, Raman peaks at 785 cm −1 (phosphodiester bonds of DNA) and 1,342 cm −1 (guanine) were more predominant in d18 stat mESC-CMs.…”

“…We chose days 10 and 20 for analyses since these time points have been previously reported to be crucial for early- and mid-stage assessment of cardiac maturation (Satin et al., 2004, Lundy et al., 2013, Brauchle et al., 2016). The differentiation efficiency using this protocol was confirmed by imaging flow cytometry (Figure S4).…”

Cardiac differentiation of pluripotent stem cells and implications for modeling the heart in health and disease

“…More recently, a label-free, noninvasive technique that identifies cardiac subtypes based on their intrinsic spectral differences identified by Raman spectroscopy has been reported (75). Although this is an innovative approach, it may not be ideal for accurate separation of hPSC-derived atrial or ventricular CMs.…”

Raman microspectroscopy for the development and screening of recombinant cell lines

“…Although the interpretation of the molecular vibrational Raman signals from complex samples is challenging, various studies have shown that Raman profiles resemble cell phenotype-specific features, which, in combination with multivariate analysis methods, enable a marker-free identification of specific cell populations and the discrimination of even very closely related cell types from each other [2]. Raman microspectroscopy, similar to wellestablished marker-based methods for cellular characterization such as flow cytometry, is capable to identify tumor cells, to detect and differentiate cell death modalities in vitro, and to monitor stem cell-derived cell populations [3][4][5]. Most of the applications of Raman microspectroscopy focus on tissue engineering, regenerative medicine or tissue diagnostics.…”