Dissecting Heterogeneity Reveals Functional Subpopulation of Stem Cells From Human Exfoliated Deciduous Teeth

Abstract

Objectives: Identify the functional subpopulation's surface marker of stem cells from human exfoliated deciduous teeth (SHEDs) using single-cell RNA sequencing (scRNA-seq) and isolate the subset, providing a theoretical basis for the refined application of stem cells.

Methods: scRNA-seq identified SHEDs as distinct subpopulations. GO and KEGG analyses were performed to define each subpopulation. Pseudotime trajectory analysis was employed to elucidate the potential differentiation processes of SHEDs. To investigate the multipotent differentiation potential of the BAMBI⁺ subpopulation, we conducted qPCR, Western blot analysis, Alizarin Red staining, Oil Red O staining, Alcian Blue staining, and immunohistochemical staining. The angiogenic capacity of the BAMBI⁺ subpopulation was also verified through cratch tests, transwell migration assays, qPCR, Matrigel tube formation assays, and chick embryo chorioallantoic membrane (CAM) assay.

Results: Major subpopulations of SHEDs, including a proliferative subpopulation, a functional subpopulation, fibroblast-like cells, were identified. As for the transformation trajectories, the proliferative subpopulation is the root population, which then differentiates into a functional subpopulation or fibroblast-like cells. The functional subpopulation is characterized by BAMBI expression and exhibits strong angiogenic, chondrogenic, and proliferative capabilities. Cells at different passages presented distinct and common characteristics.

Conclusions: SHEDs constitute a heterogeneous population and BAMBI⁺ subpopulation has higher proliferative, chondrogenic and angiogenic capacities.

Clinical relevance: This study offers a theoretical basis for optimizing SHED-based cell therapeutic strategies. BAMBI⁺ SHEDs, excelling in angiogenesis/chondrogenesis, may be used for tissue-engineered therapies of the clinical disorders such as temporomandibular joint disc regeneration via "vascularize-then-cartilage" strategy.

Keywords: BAMBI; Heterogeneity; Single-cell RNA sequencing; Stem cells from human exfoliated deciduous teeth.