231 Novel protocol for the invitro maintenance and expansion of adult epidermal LGR5+ stem cells

Abstract

Currently, the standard for treatment of full-thickness skin wounds is skin grafts or bioengineered skin substitutes; however, this method is limited by the amount of intact donor skin and lack of follicles and architecture. Thus, a protocol is needed for the expansion and differentiation of adult epidermal and hair follicle stem cells for use in scaffold mediated tissue engineering. Recently, we developed a transgenic porcine model in which H2B-GFP is under the control of the LGR5 promoter. LGR5 is an established marker of stem cells, meaning this model can be used to track the development and behaviour of these cells. The focus of this project was to create a novel culture method for the maintenance and expansion of LGR5+ epidermal adult stem cells utilising the green fluorescent protein (GFP) tag. Single cell epidermal stem cells were isolated from porcine skin using dispase II (10mgmL−1; Sigma) and trypsin (0.05%; Corning). Porcine fetal fibroblasts (PFF) or mouse embryonic fibroblasts (MEF) were grown to 95% confluence in a 6-well plate. Feeder layer cells were mitotically inactivated by incubation with mitomycin C (Sigma Aldrich, 10μgmL−1). Three different media were tested: basal medium [Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, Corning; Ham's F12, ThermoFisher; fetal bovine serum, Gemini Bio-Products], basal media + 5-azacytidine (Sigma Aldrich) and CHIR99021 (Tocris), or basal media + keratinocyte growth supplements (transferrin, hydrocortisone, T3, adenine, insulin, cholera toxin; Sigma Aldrich, epidermal growth factor; R&D Systems). Epidermal cells were plated in each medium for both PFF and MEF feeder layers. Experiments were performed in technical duplicates and replicated 3 times. On Day 9, total numbers of colonies in each well were counted and number of GFP-positive cells were quantified using ImageJ (National Institutes of Health). Results in Table 1 show that overall, the MEF feeder layer was able to support a higher rate of growth (P<0.05) and maintain the LGR5+ lineage at a higher proportion under all of the experimental conditions (P<0.05). In the growth-supplemented media, MEFs had fewer colonies than PFFs, but MEF colonies were, on average, 2.5 times larger (P<0.05). Conditions containing 5-aza and CHIR were the only conditions to maintain the LGR5+ lineage on the feeder layer. Statistically significant differences (P<0.05) were determined using two-way ANOVA, followed by Tukey's HSD test. Next, LGR5+ cells will be plated in media containing additional growth factors to stimulate expansion, while using CHIR and 5-aza to maintain the LGR5+ lineage. This protocol could be used in scaffolds to create three-dimensional growth of skin invitro and lead to better grafts for burn victims. Table 1.Growth of LGR5+ cells in different media including 5-azacytidine (5-aza), CHIR 99021 (CHIR), and keratinocyte growth supplements Group1 Basal medium (BM) BM + 5-aza+ CHIR BM + growth supplements No. of colonies/well MEF 127.7±40.8AB 189.3±16.9A 87.3±14.6B PFF 65.0±14.1A 83.3±17.0AB 148±33.7B Average no. of GFP+ cells per frame MEF 0.5±0.8B 65.7±18.4A 1.8±1.7B PFF 0.9±1.0B 22.6±4.5A 0.3±0.6B A,BValues within rows with different superscripts differ (P ≤ 0.05). 1MEF=mouse embryonic fibroblasts; PFF=porcine fetal fibroblasts.