Oral Presentation ASDR-AWTRS-MEPSA 2018 Joint Meeting

Background: The current gold standard treatment for large skin deficits such as severe burns or blistering diseases is autologous transplantation of a patient’s own keratinocytes, following ex-vivo expansion on a murine Swiss 3T3-J2 cell feeder layer. Problem: <1% of harvested patient keratinocytes are recruited in culture, resulting in prolonged hospitalisation periods prior to transplantation with an increased risk of  infection and mortality. This, combined with the use of xenogeneic feeder cells and culture reagents, leaves considerable room for the improvement of patient keratinocyte culture. Hypothesis: We have previously demonstrated that pericytes promote epidermal renewal in in vitro organotypic cultures by increasing planar keratinocyte cell divisions – thus facilitating retention of cells within the proliferative compartment. We hypothesise that pericytes may be a better feeder layer than Swiss 3T3s capable of improving both seeding efficiency and keratinocyte self-renewal in vitro.  Method & Results: We optimised methods to generate a mitotically inactive pericyte feeder layer, and tested its ability to support clonal expansion of freshly isolated primary keratinocytes from human neonatal foreskin tissue. Our data show that dermal pericytes promote greater keratinocyte colony forming efficiency than Swiss 3T3-J2 feeders (p=0.0011) and to an even greater extent than dermal fibroblasts (p>0.0001). Moreover, pericytes promoted the formation of larger colonies compared to both fibroblasts and Swiss 3T3-J2 cells. Current experiments are aimed at investigating whether all basal keratinocytes are equally stimulated to grow on a pericyte feeder layer or if the effects are specific for keratinocyte subsets i.e. the α₆^brightCD71^dim keratinocyte stem cell, the α₆^brightCD71^bright transit amplifying or the α₆^dim early-differentiating populations. Conclusion: These data suggest that pericytes may be a valid substitute for murine Swiss 3T3-J2 cells and provide a first step towards developing alternative approaches to treating patients with severe skin deficits.