Human Embryonic Stem Cells: An Alternative Source for Clinical-grade Keratinocyte

Objectives: Autologous keratinocytes are supposed to be ideal for epidermal grafts. However, it takes 3-4 weeks to get enough cells, which may put patient’s health iat the risk due to their limited availability in real time. Recently Human embryonic stem cells (hESCs), which are pluripotent by the nature and can be differentiated spontaneously into all three germ layers, have been proposed as an alternative source for keratinocyte to treat patients in real time without any delay. However, current strategies involve getting hESCs derived keratinocyte (hESCs-Kert) are hampered by the use of xenogeneic microenvironment which limits their clinical application. Therefore, it would be crucial to eliminate or at least reduce the xenogeneic components to increase the clinical application of hESCs- Kert. 

Methods: To achieve our goal we made step wise strategy. Step 1: H1-hESCs were first differentiated into fibroblast (H1-ebF). Step 2: H1-hESCs were co-cultured with inactivated H1-ebF to establish an autogenic microenvironment (ACC system). Step 3: H1-hESCs were derived into keratinocyte lineage (H1-Kert) in an ACC system in the presence of Retinoic Acid (RA) and Activin. Characterization of H1-Kert was done at mRNA (Reverse transcription polymerase chain reaction (RT-PCR), real time RT-PCR) and protein level (flow cytometry analysis and immunofluorescence technique) at different time points.

Results: Quantitative characterization of H1-Kert showed the progressive increase of keratinocyte markers (P63 and K14) with the passage of time. Concurrently, we found the progressive reduction in pluripotency marker (Nanog). We were able to get relatively pure population of keratinocyte (K14: 72%; P63: >90%; Alpha Integrin 6: 72%) by Day 30. 

Conclusions: We described a successful differentiation of H1-hESCs into keratinocyte with the minimum use of animal source. This research is a step forward towards a xeno-free differentiation of hESCs progenitors for their potential clinical application