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

Abstract

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 is well established, which influences their regenerative and therapeutic potentials. As highly proliferative/multipotent DPSCs are minority subpopulations within dental pulp, the development of noninvasive strategies, capable of successfully discriminating between DPSC subpopulations 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 subpopulations with contrasting proliferative and differentiation capabilities isolated from dental pulp tissues. Individual DPSC subpopulations were isolated from human third molars and identified as high or low proliferative and multipotent or unipotent, following in vitro expansion and senescence confirmation. High proliferative/multipotent DPSCs, such as A3 (18 population doublings [PDs] and 60PDs), and low proliferative/unipotent DPSCs, including A1 and B1 (8PDs and 7PDs, respectively), were analyzed 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 subpopulation (10 spectra per nuclear and 10 spectra per cytoplasmic/membrane region in each cell analyzed), over 500–2100 cm⁻¹. Spectra and peak assignments were obtained, followed by principal component analysis (PCA) and multivariate statistical analysis. Although DPSC spectra contained typical Raman peaks for nucleic acids, proteins, and lipids, the spectral intensities of high proliferative/multipotent 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/multipotent DPSCs, A3 at 18PDs and 60PDs; versus the similar overlapping profiles for low proliferative/unipotent DPSCs, A1 (8PDs) and B1 (7PDs). This study confirms that SCRM successfully discriminates between DPSC subpopulations with contrasting proliferative and differentiation capabilities, advocating its further assessment as a viable technique for the selective noninvasive screening, identification, and isolation of high proliferative/multipotent DPSCs from dental pulp tissues for regenerative medicine applications.

Impact Statement

This study is the first to investigate and confirm the effectiveness of single-cell Raman spectroscopy (SCRM), in its ability to discriminate between dental pulp stem cells (DPSCs) with contrasting proliferative and differentiation capabilities. The findings show that SCRM can rapidly and noninvasively distinguish and identify DPSC subpopulations in vitro with superior proliferative and multipotency properties, versus lesser quality DPSCs, thereby overcoming the significant heterogeneity issues surrounding DPSC ex vivo expansion and differentiation capabilities. Such findings support further SCRM assessment for the selective screening/isolation of superior quality DPSCs from whole dental pulp tissues, for more effective in vitro evaluation and therapy development.