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

Minxiong Li,Jun Ma,Yuchen Li,Zhending She,Lei Yang,Mengru Dong,Yanbin Gao,Zijun Zheng,Rongwei Tan

doi:10.1186/s13287-020-01645-3

Minxiong Li, Jun Ma + Show 7 more

Open Access

https://doi.org/10.1186/s13287-020-01645-3

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Abstract

BackgroundThree-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.Methods3D 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).ResultsTypical 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.ConclusionsCSS is positive to promote epithelial differentiation of hASCs, which will foster a deeper understanding of artificial dermis in skin wound healing and regeneration.

Highlights

Three-dimensional (3D) cultivation with biomaterials was proposed to facilitate stem cell epithelial differentiation for wound healing
Characterization of human adipose-derived stem cells (hASCs) Frozen hASCs of 3rd passage (P3) successfully adhered to petri dishes after recovery and reached > 90% confluency when cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) 3 days later
According to the flow cytometry analysis, the hASCs were shown to be positive for cell surface marker CD90 and CD44, but a negative or low expression for CD11b, CD34, CD45, and HLADR markers (Fig. 2b)

Summary

Introduction

Three-dimensional (3D) cultivation with biomaterials was proposed to facilitate stem cell epithelial differentiation for wound healing. Whether human adipose-derived stem cells (hASCs) on collagen sponge scaffold (CSS) better differentiate to keratinocytes remains unclear. Wound healing is a complex multi-step process that mainly experiences four continuous phases, namely hemostasis, inflammation, proliferation, and maturation or remodeling [6, 7]. Skin re-epithelialization is a crucial step in the proliferation phase [10, 11], of which keratinocytes, the main components of the epidermis of the skin, play an important role in promoting re-epithelialization [4, 11]. The keratinocytes from the edges of the wound begin to migrate towards the center within hours after injury and proliferate continuously until the epithelial surface is intact [12]. Keratinocytes require specific culture conditions and become senescent after only 10–20 doublings [15], which hampers the production of sufficient cells for graft [16]

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