The corneal epithelial layer is continuously replaced by limbal stem cells. Reconstructing this layer in vitro using synthetic scaffolds is highly needed. Poly-lactic-co-glycolic acid (PLGA) is approved for human use due to its biocompatibility and biodegradability. However, PLGA is hydrophobic, preventing cell adherence to PLGA membranes. PLGA scaffolds were prepared by electrospinning on a custom-made target drum spinning at a rate of 1000 rpm with a flow rate of 0.5 mL/h and voltage at 20 kV, then treated with oxygen plasma at 30 mA using a vacuum coater. Scaffolds were characterized by SEM, mechanically by tensile testing, and thermally by DSC and TGA. In vitro degradation was measured by weight loss and pH drop. Wettability was assessed through water uptake and contact angles measurements. Human limbal stem cells (hLSCs) were isolated and seeded on the scaffolds. Cell attachment and cytotoxicity assay were evaluated on day 1 and 5 after cell seeding. SEM showed regular fiber morphology with diameters ranging between 150 nm and 950 nm. Tensile strength demonstrated similar average stress values for both plasma- and non-plasma-treated samples. Scaffolds also showed gradual degradability over a period of 7–8 weeks. Water contact angle and water absorption were significantly enhanced for plasma-treated scaffolds, indicating a favorable increase in their hydrophilicity. Scaffolds have also supported hLSCs growth and attachment with no signs of cytotoxicity. We have characterized a nanofiber electrospun plasma-treated PLGA scaffold to investigate the mechanical and biological properties and the ability to support the attachment and maintenance of hLSCs.

The stemness in keratinocyte stem cells (KSCs) is determined by their gene expression patterns. KSCs are crucial in maintaining epidermal homeostasis and wound repair and are widely used candidates for therapeutic applications. Although several studies have reported their positive identifiers, unique biomarkers for KSCs remain elusive. Here, we aim to identify potential candidate stem cell markers. Human epidermal keratinocytes (HEKs) from neonatal foreskin tissues were isolated and cultured. Single-cell clonal analysis identified and characterized three types of cells: KSCs (holoclones), transient amplifying cells (TACs; meroclones), and differentiated cells (DSCs; paraclones). The clonogenic potential of KSCs demonstrated the highest proliferation potential of KSCs, followed by TACs and DSCs, respectively. Whole-transcriptome analysis using microarray technology unraveled the molecular signatures of these cells. These results were validated by quantitative real-time polymerase chain reaction and flow cytometry analysis. A total of 301 signature upregulated and 149 downregulated differentially expressed genes (DEGs) were identified in the KSCs, compared to TACs and DSCs. Furthermore, DEG analyses revealed new sets of genes related to cell proliferation, cell adhesion, surface makers, and regulatory factors. In conclusion, this study provides a useful source of information for the identification of potential SC-specific candidate markers.