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. 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). 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. CSS is positive to promote epithelial differentiation of hASCs, which will foster a deeper understanding of artificial dermis in skin wound healing and regeneration.

中文翻译：

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通过间接共培养策略，使用胶原海绵支架（CSS）进行三维（3D）培养的人类脂肪干细胞（hASCs）的上皮分化。

提出了用生物材料进行的三维（3D）培养，以促进干细胞上皮细胞的分化，从而促进伤口愈合。然而，胶原海绵支架（CSS）上的人类脂肪干细胞（hASC）是否能更好地分化为角质形成细胞尚不清楚。将在hASC表皮分化上与HaCaT细胞在气液界面（ALI）上共培养的CSS进行的3D培养与二维（2D）形式进行了比较，并在没有“共培养”或“ ALI”的情况下进行了培养。评估细胞形态，细胞粘附和生长条件，然后评估角蛋白14（K14，角质形成细胞特异性标志物）的蛋白质和基因表达。在ALI共培养的hASC中，观察到了典型的角质形成细胞的鹅卵石形态。但是在倒置显微镜和扫描电子显微镜下，那些接种在CSS上的细胞显示出更多的角质形成细胞样细胞。罗丹明标记的鬼笔环肽染色在3D分化组中证实了所需的细胞粘附和增殖，与H＆E染色一致。与在2D培养系统中培养或未使用“ ALI”的培养相比，免疫荧光染色和基因表达分析显示，在CSS上共培养的hASC在第15天时以较高的K14表达K14。CSS可以促进hASC的上皮分化。深入了解人造真皮在皮肤伤口愈合和再生中的作用。