Discrimination of Dental Pulp Stem Cell Regenerative Heterogeneity by Single Cell Raman Spectroscopy.

Dental pulp stem cells (DPSCs) are increasingly being recognized as a viable cell source for regenerative medicine. However, significant heterogeneity in their ex vivo expansion capabilities are well-established, which influences their regenerative and therapeutic potentials. As highly proliferative/multi-potent DPSCs are minority sub-populations within dental pulp, the development of non-invasive strategies capable of successfully discriminating between DPSC sub-populations with contrasting proliferative and differentiation capabilities in situ, would be immensely beneficial for the selective screening/isolation of superior quality DPSCs for in vitro assessment and therapy development. Consequently, this study assessed the effectiveness of Single Cell Raman Spectroscopy (SCRM), in distinguishing between DPSC sub-populations with contrasting proliferative and differentiation capabilities isolated from dental pulp tissues. Individual DPSC sub-populations were isolated from human third molars and identified as high or low proliferative and multi-potent or uni-potent, following in vitro expansion and senescence confirmation. High proliferative/multi-potent DPSCs, such as A3 (18PDs and 60PDs) and low proliferative/uni-potent DPSCs, including A1 and B1 (8PDs and 7PDs respectively), were analysed using an iHR550 Raman Spectrometer, equipped with a CCD camera and Eclipse Ti-U Inverted Microscope. Single cell spectra were acquired for 20 cells in each sub-population (10 spectra per nuclear and 10 spectra per cytoplasmic/membrane regions in each cell analysed), over 500-2100 cm-1. Spectra and peak assignments were obtained, followed by PCA and multivariate statistical analysis. Although DPSC spectra contained typical Raman peaks for nucleic acids, proteins and lipids, the spectral intensities of high proliferative/multi-potent DPSCs, A3 (18PDs), were higher than A3 (60PDs), A1 (8PDs) and B1 (7PDs), reflecting significantly elevated DNA (729 cm-1) and protein (1111, 1167, 1245 and 1680 cm-1) contents overall. PCA and multivariate analysis revealed significant variations in scatter plots and Raman signatures, with distinct fingerprints for high proliferative/multi-potent DPSCs, A3 at 18PDs and 60PDs; versus the similar overlapping profiles for low proliferative/uni-potent DPSCs, A1 (8PDs) and B1 (7PDs). This study confirms that SCRM successfully discriminates between DPSC sub-populations with contrasting proliferative and differentiation capabilities, advocating its further assessment as a viable technique for the selective non-invasive screening, identification and isolation of high proliferative/multi-potent DPSCs from dental pulp tissues for regenerative medicine applications.