Keratinocyte differentiation of hESCs in a novel autogenic co-culture system

Introduction: Differentiation of human embryonic stem cells (hESCs) into keratinocytes holds a great potential for future dental applications. Current protocols with the use of xenogenic components for keratinocyte differentiation and propagation limits the potential clinical application. Objectives: To induce differentiation of keratinocytes from hESCs in a novel autogenic system. Materials and Methods: For autogenic co-culture system fibroblast were differentiated from the hESCs through EB formation and further differentiation on 0.1% gelatin. For keratinocyte differentiation, hESCs were seeded on inactivated hESCs derived fibroblast with Define Keratinocyte Serum Free Medium (D-KSFM), which contained retinoic acid and activin. After 7 days, these hESCs progenies were detached and cultured on human Type I collagen for further differentiation. Characterizations of the differentiated cells were done by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time RT-PCR and immunofluorescence staining. Results: Differentiated cells at low density formed holoclones, meraclones and paraclones. At high density they formed a polarized mono-layer. They also expressed keratinocyte markers K14, P63 and K18 at mRNA and protein level. Differentiated cells were compared with negative control. K14 expression was found to be 1.8-fold higher in autogenic co-culture system than the feeder-free matrigel system (p< 0.05). Conclusions: This study brings a novel knowledge that autogenic system can be used for keratinocyte differentiation from hESCs. The present study highlights the possibility of developing a novel model system for keratinocyte differentiation with reduced use of xenogenic sources.

This work was partially supported by grants from the Ministry of Education of Singapore (R223000014112 and R223000018112).