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/regenerative DPSCs are minority sub-populations within dental pulp, the development of non-invasive strategies capable of successfully discriminating between high and low proliferative/regenerative DPSC sub-populations 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 high and low proliferative/regenerative DPSC sub-populations isolated from dental pulp tissues. Individual DPSC sub-populations were isolated from human third molars and identified as high or low proliferative/regenerative, following in vitro expansion and senescence confirmation. High proliferative/regenerative DPSCs, such as A31 (18PDs and 60PDs) and low proliferative/regenerative DPSCs, including A11 and B11 (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 cytoplasmic/membrane regions), over 600-1800 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/regenerative DPSCs, A31 (18PDs), were higher than A31 (60PDs), A11 (8PDs) and B11 (7PDs), reflecting significantly elevated DNA (729 cm-1) and protein (1167, 1245, 1629 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/regenerative DPSCs, A31 at 18PDs and 60PDs; versus the similar overlapping profiles for low proliferative/regenerative DPSCs, A11 (8PDs) and B11 (7PDs). This study confirms that SCRM successfully discriminates between high and low proliferative/regenerative DPSC sub-populations, advocating its further assessment as a viable technique for the selective non-invasive screening, identification and isolation of high proliferative/regenerative DPSCs from dental pulp tissues for regenerative medicine applications.