ABSTRACT
Fibroblast heterogeneity plays a central role in wound healing and fibrosis, yet the mechanisms driving transitions between fibroblast subpopulations remain poorly understood. CD90 (THY1) is a surface marker linked to extracellular matrix remodelling and fibrotic activity. We hypothesize that CD90⁺ and CD90⁻ fibroblast states represent dynamic phenotypes rather than fixed lineages, and that non-canonical DNA structures (G-quadruplexes and i-Motifs) may act as molecular switches regulating these transitions. We employed Fluorescence-Activated Cell Sorting (FACS) to isolate CD90⁺ and CD90⁻ fibroblast subpopulations based on CD90 expression. Initial analysis identified CD90+ (~90%) and CD90- (~10%) populations. To examine population stability and functional dynamics, we cultured and monitored three experimental conditions: sorted CD90⁺, sorted CD90⁻, and unsorted mixed populations. CD90 expression and collagen (COL1) deposition were evaluated over time using dual immunocytochemistry. To capture transcriptional heterogeneity underlying subpopulations, 5’-single-cell RNA sequencing (scTECH-seq) was performed on multiplexed samples labelled with short barcoded oligonucleotides (SBOs). Following demultiplexing using D-score algorithm, data were integrated and clustered using Seurat v5 to define subpopulation-specific gene expression profiles. Time-course analysis revealed dynamic population reversion in sorted cultures. Both sorted CD90+ and CD90- populations progressively shifted towards the original CD90+/CD90- ratio over time, demonstrating inherent plasticity. Unsorted cultures maintained stable ratios, indicating an intrinsic regulatory balance. Dual CD90/collagen staining showed distinct temporal patterns: CD90⁺ cells deposited more collagen, although this relationship shifted dynamically during phenotypic reversion. scRNA-seq analysis revealed differential gene expression between subpopulations. CD90+ clusters exhibited elevated expression of extracellular matrix genes (COL1A1, COL3A1, FN1) and TGF-β responsive pathways, consistent with their collagen-rich, fibrogenic phenotype. The dynamic reversion of CD90+/- populations highlights the inherent plasticity of fibroblast subpopulations. Integrating single-cell transcriptomics with non-canonical DNA structure biology will further elucidate how chromatin architecture may influence fibroblast fate, offering novel molecular targets to modulate fibrosis and improve regenerative outcomes.