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

Area of Science:

• Stem Cell Biology
• Regenerative Medicine
• Genomics

Background:

• Stem cells from human exfoliated deciduous teeth (SHEDs) are a promising source for regenerative therapies.
• SHEDs are known to be a heterogeneous cell population.
• Understanding SHED heterogeneity is crucial for optimizing their therapeutic applications.

Purpose of the Study:

• To identify the specific surface marker of the functional subpopulation within SHEDs using single-cell RNA sequencing (scRNA-seq).
• To isolate this functional subpopulation and characterize its properties.
• To provide a theoretical foundation for the refined use of SHEDs in cell-based therapies.

Main Methods:

• Single-cell RNA sequencing (scRNA-seq) to identify distinct SHED subpopulations.
• Gene Ontology (GO) and KEGG pathway analyses to define subpopulation characteristics.
• Pseudotime trajectory analysis to understand differentiation pathways.
• Functional assays including qPCR, Western blot, Alizarin Red, Oil Red O, Alcian Blue staining, immunohistochemistry, angiogenesis assays (cratch tests, transwell, Matrigel, CAM assay) to evaluate the BAMBI-positive subpopulation.

Main Results:

• scRNA-seq revealed major SHED subpopulations: proliferative, functional, and fibroblast-like cells.
• The proliferative subpopulation serves as the root, differentiating into functional or fibroblast-like cells.
• The functional subpopulation is characterized by BAMBI expression and demonstrates significant angiogenic, chondrogenic, and proliferative capabilities.
• Distinct and common characteristics were observed across different cell passages.

Conclusions:

• SHEDs represent a heterogeneous cell population.
• The BAMBI-positive subpopulation exhibits superior proliferative, chondrogenic, and angiogenic potential.
• BAMBI-positive SHEDs are suitable for tissue-engineered therapies, particularly for conditions like temporomandibular joint disc regeneration, utilizing a 'vascularize-then-cartilage' approach.