Epithelial differentiation of human adipose-derived stem cells (hASCs) undergoing three-dimensional (3D) cultivation with collagen sponge scaffold (CSS) via an indirect co-culture strategy

Background Three-dimensional (3D) cultivation with biomaterials was proposed to facilitate stem cell epithelial differentiation for wound healing. However, whether human adipose-derived stem cells (hASCs) on collagen sponge scaffold (CSS) better differentiate to keratinocytes remains unclear. Methods 3D cultivation with CSS on hASC epidermal differentiation co-cultured with HaCaT cells at air-liquid interface (ALI) was compared with two-dimensional (2D) form and cultivation without co-culture or ALI. Cellular morphology, cell adhesion, and growth condition were evaluated, followed by the protein and gene expression of keratin 14 (K14, keratinocyte specific marker). Results Typical cobblestone morphology of keratinocytes was remarkably observed in co-cultured hASCs at ALI, but those seeded on the CSS exhibited more keratinocyte-like cells under an invert microscope and scanning electron microscope. Desired cell adhesion and proliferation were confirmed in 3D differentiation groups by rhodamine-labeled phalloidin staining, consistent with H&E staining. Compared with those cultured in 2D culture system or without ALI, immunofluorescence staining and gene expression analysis revealed hASCs co-cultured over CSS expressed K14 at higher levels at day 15. Conclusions CSS is positive to promote epithelial differentiation of hASCs, which will foster a deeper understanding of artificial dermis in skin wound healing and regeneration.