(745c) Identifying the Differentiation and Proliferation Stage of Single Hematopoietic Stem Cells Using Raman Microspectroscopy

The ability to monitor the differentiation and proliferation status of individual hematopoietic stem cells is critical to understanding the effects of various external factors, such as matrix properties and cytokines, on hematopoiesis.  Raman microspectroscopy is a promising method for single cell identification and characterization. Unlike other invasive methods, Raman microscopy has the ability to detect changes in the composition of living cells without compromising the cells’ morphology, proliferation, or pluripotency. However, identifying cells according to their Raman spectra is difficult due to a lack of spectral bands that are unique to a single cell line. Here, we report our ability to identify the differentiation stage of individual mouse hematopoietic stem cells and mature cells by multivariate analysis of their Raman spectra.  Confocal Raman microscopy was used to obtain spectral fingerprints of single cells in various stages of hematopoiesis. A model of the cell type-specific variance in the Raman spectra was constructed using partial least-squares discriminant analysis (PLS-DA) and enabled accurately identifying cells of “unknown” differentiation stage, including closely-related long- and short-term hematopoietic stem cells with low prediction error (<10%). Our high identification accuracy cannot be attributed to spectral contributions from the polyacrylamide gel substrates, which varied in stiffness and thickness, and culture media. In addition, principal component analysis was used to identify the cell cycle stage of each single cell based on the principal component corresponding to DNA-related Raman peaks. Stem cells were found to be more proliferative than mature cells. Noteworthy, the Raman contributions that correlated to cell cycle phase did not significantly interfere with identification of cell differentiation stage. This method allows for non-invasive monitoring of stem cell differentiation over time with location specificity, and can be used to correlate external stimuli with stem cell fate decisions.