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

• Published in: Analytical chemistry (Washington) Vol. 84; no. 7; pp. 3155 - 3162
• Main Authors: Ghita, Adrian, Pascut, Flavius C, Mather, Melissa, Sottile, Virginie, Notingher, Ioan
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 03.04.2012
• Subjects: Analytical chemistry; Animals; Cell Differentiation; Cell Survival; Chemistry; Cytoplasm; Cytoplasm - metabolism; Discriminant Analysis; Exact sciences and technology; Fluorescence; General, instrumentation; Mice; Multivariate Analysis; Neural Stem Cells - cytology; Neuroglia - cytology; Phenotype; Principal Component Analysis; Principal components analysis; Ribonucleic acid; RNA; RNA - metabolism; Spectrometric and optical methods; Spectrum Analysis, Raman - methods; Stem cells; Staining; Fluorescence; Instrumentation; Repression; Linear model; Fluorescence spectrometry; Characterization; Specificity; Messenger RNA; Detection limit; Raman spectrometry; Nucleic acid; Monitoring; Discriminant analysis; Use; Concentration; Calibration; Marker; Real time; In vitro; Sensitivity; Volume; Technique; Differentiation; Principal component analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac202994e

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.