Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status

Abstract

Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ~700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (~1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA.

Raman spectroscopy: techniques and applications in the life sciences

“…Although near-infrared lasers are preferred in Raman spectroscopy of live cells, the choice of wavelength, power and illumination conditions need to be assessed according to the specific biological processes and type of cells investigated. For example, the use of staining tests (Trypan Blue, dead/live kit based on SYTO10 dye and ethidium homodimer-2) has been used to evaluate viability of cells after Raman analysis [190,191]. Time-course Raman experiments (785 nm laser, <100 mW, 5-10 min illumination, 60x/1.2 NA objective) monitoring the interaction of individual Toxoplasma gondii with host human cells indicated no differences in infection potential, whether or not the parasites were analysed by Raman spectroscopy or not [145].…”

Methods and Applications of Raman Microspectroscopy to Single-Cell Analysis

“…73 In extensive work further analyzing the distinctive differences between human stem cells and their derivatives, Konorov et al [74][75][76] were able to show a major discriminant between the different cell types is their glycogen content. In very recent work, Ghita et al 77 investigated cytoplasmic RNA in undifferentiated neural stem cells (NSC) as a potential marker to assess their degree of differentiation. By applying LDA, Ghita and colleagues were able to discriminate between differentiated and undifferentiated NSC with a sensitivity of 89.4% and specificity of 96.4%.…”

Analysis of interaction between the apicomplexan protozoan Toxoplasma gondii and host cells using label-free Raman spectroscopy

“…RMS is a well-established analytical technique that enables label-free chemical analysis of individual cells and bacteria with sub-micrometric spatial resolution. [4][5][6][7][8] RMS was also used for time-course experiments on individual live cells revealing molecular processes not attainable with other imaging. [9][10][11][12][13] The integration of environmental chambers with inverted Raman microscopes can allow time-and spatially-resolved molecular studies of cellular processes spanning days and weeks, such as stem cell differentiation.…”