Optimisation of Culture Conditions for Expansion of Human Epidermal Stem Cells

Objectives The present study attempted to optimise in vitro culture methods for establishing a homogeneous population of human epidermal stem cells (EpSCs). Material and Methods Human skin samples were obtained with informed written consent taken priorly to. The isolation and expansion of EpSCs was performed by explant and suspension methods using different culture media (Minimum essential medium (MEM α) and EpiLife) with supplementation of foetal bovine serum (FBS at 0, 10, and 20%) and growth factors [2 ng/mL keratinocyte growth factor, (KGF) and 2 ng/mL epidermal growth factor (EGF), 1% human keratinocyte growth supplement (HKGS)]. EpSCs were analysed for distinct biological features, such as self-renewal ability, cell cycle status, and cytogenetic stability. Further, the cells were quantitatively assessed for stem cell and differentiation marker expressions. Results Among the culture methods, explant culture showed the attachment of cells in MEM-α at 20% FBS supplementation by exhibiting a typical “cobblestone” epithelial pattern of growth at passage 0, but did not retain a similar morphology at passage 1. In contrast, no cells were firmly attached in suspension culture, either in MEM-α, at all concentrations of FBS. Interestingly, a higher number of proper EpSCs from suspension culture was observed when MEM-α was supplemented with FBS and growth factors, such as KGF, EGF, and HKGS. In EpiLife media with HKGS, the primary culture did not support the attachment of cells. Whereas, the EpiLife media supplemented with KGF, EGF, and HKGS resulted in the correct attachment of EpSCs with typical morphological features. EpSCs established from MEM-α and EpiLife supplemented with growth factors were proliferative and had more cells in the quiescent phase of the cell cycle. The analyses showed that EpSCs had high clonogenic potential with varied levels of expression of selected markers, such as p63, SCA1, keratin 5 (KRT5), KRT14, KRT15, ITGA6, ITGB1, KRT1, KRT10, and filaggrin (FLG). Conclusion The study findings indicated that EpSCs established in MEM-α and EpiLife media with growth factors possess plasticity with high proliferation ability. Thus, EpSCs may represent an ideal source for skin regenerative medicine applications.