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In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics
Chee-Wai Wong1,2, Catherine F LeGrand1,2, Beverley F Kinnear1,2
1School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, WA, 6102, Australia.
Scientific Reports
|December 8, 2019
Summary
This study developed a xenogeneic-free culture system to expand human keratinocytes, maintaining their stem-like state and enhancing proliferation for potential clinical applications in skin regeneration.
Area of Science:
- Stem Cell Biology
- Tissue Engineering
- Dermatology
Background:
- Long-term expansion of keratinocytes without animal products often leads to reduced proliferation and increased differentiation.
- The extracellular matrix (ECM) is crucial for cell fate determination in vivo.
- Existing methods for xenogeneic-free keratinocyte expansion face challenges in maintaining self-renewal capacity.
Purpose of the Study:
- To develop a xenogeneic-free culture system that preserves the self-renewal capacity of primary human keratinocytes.
- To investigate the role of human dermal fibroblast-derived ECM in supporting keratinocyte expansion.
- To evaluate the potential clinical utility of keratinocytes expanded under these conditions.
Main Methods:
- Cultured human dermal fibroblasts under macromolecular crowding to promote ECM deposition and organization.
- Produced xenogeneic-free ECM using Phospholipase A2 decellularization, analyzing its composition via proteome analysis.
- Expanded primary human keratinocytes on the prepared ECM and assessed proliferation, differentiation markers (p63, keratin 10, keratin 16), and colony-forming efficiency.
Main Results:
- Keratinocytes cultured on fibroblast-derived ECM showed rapid proliferation, retained small size, expressed p63, and lacked keratin 10 and keratin 16.
- Colony-forming efficiency was significantly enhanced on the engineered ECM compared to collagen I, indicating maintenance of a stem-like state.
- Multi-layered, strong, and stable keratinocyte sheets were formed, superior to those grown on collagen I.
Conclusions:
- The developed xenogeneic-free culture system effectively maintains human keratinocytes in a stem-like state.
- Dermal fibroblast-derived ECM supports robust keratinocyte expansion with enhanced self-renewal capacity.
- The stratified epidermal sheets produced have significant potential for clinical applications in regenerative medicine.
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