The Ex-Vivo Assembly and Transplantation of a LGR6+ Epithelial Stem Cell-Derived Hybrid Graft Using Common Acellular Matrices To Augment Wound Healing and Nascent Hair Growth in Tissues Devoid of Adnexal Structures

Denver Lough^1,2, Damon Cooney^1,2, Shaun Mendenhall^1,2, Mei Yang^1,2, Joel Reichensperger^1,2, Lisa Cox^1,2, Nicole Cosenza^1,2, Nathan Wetter^1,2, Carrie Harrison^1,2, Michael Neumeister^1,2

¹Surgery/Plastic Surgery, SIU School of Medicine, Springfield, IL, United States; ²Plastic and Reconstructive Surgery, The Johns Hopkins Hospital, Baltimore, MD, United States

Background: Previous research shows that the migration of the Leucine-rich repeat-containing G-protein coupled Receptor epithelial stem cell (LGR5+ and LGR6+ ESC) into wounds augments healing and permits nascent hair growth in areas devoid of these cells following local full thickness soft tissue trauma. Here, we assess the LGR-expressing cell’s potential as a hybrid graft in full thickness soft tissue wounds with the goal of developing an easily assembled biological dressing to provide patients with their own focal epithelial stem cell population, capable of growing functional, hair producing skin.

Methods: GFP expressing LGR6+ ESCs were isolated from C57BL/6(UBC-GFP) mice using FACS gated on CD34, CD73, and LGR6. These cells were then seeded on a variety of acellular matrices. Viability and attachment was assessed using confocal, scanning electron microscopy and bioluminescence. LRG6+ ESC seeded matrices were placed into acute 3rd degree burn wound beds of Nu/Nu mice and monitored for viability, wound healing, and growth factor expression. Bioluminescent imaging supported LGR6+ ESC confluence within the graft as well as transplant viability. Healing rates were documented and compared between mice receiving the LGR6+ ESC enriched grafts vs. control. On days 0, 5, 10 and 15, grafted areas were harvested and assessed for gross, microscopic and molecular healing using digital imaging, immunofluorescence, RT-PCR, and angiogenic proteome array analysis.

Results: LGR6+ seeded matrices show significantly enhanced gross wound healing and up regulation of key WNT, EGF, VEGF and angiogenesis mRNA transcripts and peptides, when compared to matrix-only and negative controls. Additionally, those full thickness wound beds receiving the LGR6+ ESC hybrid grafts were capable of growing nascent hair from the wound bed within a Nu/Nu murine model.

Conclusion: Here, we suggest a novel role for an LGR6+ ESC-enriched hybrid graft for the development of a fully transplantable living matrix for use in tissue engineering and reconstructive transplantation. Furthermore, with the epithelium being a major source of alloantigenicity, this system can be used to develop a series of chimeric epithelial expansion foci for engraftment within more complex composite tissue allografts in order to promote tolerance.