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Development of a Chimeric Free Flap Allograft Using the LGR6+ Epithelial Stem Cell
Denver M. Lough, MD, Ph.D1, Damon Cooney, MD, Ph.D2, Shaun Mendenhall, MD3, Joel Reichensperger, BS3, Lisa Cox, BS3, Nicole Cosenza, MS3, Nathan Wetter, MD3, Carrie Harrison, BS3, Michael Neumeister, MD3.
1SIU School of Medicine/Johns Hopkins, Springfield, IL, USA, 2Johns Hopkins, Baltimore, MD, USA, 3SIU School of Medicine, Springfield, IL, USA.
Reconstructive Transplantation (RT) and Reconstructive Microsurgery have seen enormous growth over the last decade with regards to both surgical technique and post operative monitoring and therapy. Understanding the nature of the chronic cellular rejection cascade seen in RT, in that it primarily initiates with the epithelium, we seek to apply our knowledge of the LGR epithelial stem cell (ESC) system, an epithelial stem cell capable of developing the skin, hair shaft and sweat gland, in developing the first chimeric free flap allograft. From this LGR recipient seeded chimeric allograft free flap, we intend to determine if the slow repopulation of the recipient epithelium from its own autologous stem cell niche, while in the presence of donor tissues, can lead to more successful transplantation as well as enhanced tolerance and freedom from rejection.
Using populations of age matched Sprague Dawley and Lewis rat species within a DIEP based abdominal wall transplant model, we transplanted a donor partially de-epithelialized (reticular dermis) composite tissue allograft containing abdominal wall to a recipient abdominal wall defect. Following inset of the flap, a recipient-derived LGR GFP+ ESC seeded acellular matrix was grafted onto the flap’s exposed dermis and bolstered in place. Recipients received either LGR6+ ESC graft, standard skin grafts and either rapamycin-based mono-therapy or normal saline as control. Specimens were examined for histological signs of rejection using published protocols as well as additional assays utilizing: immunofluorescent up-regulation of apoptosis, tunnel assay augmentation and recipient T-cell infiltrate. Additionally high-throughput gene arrays examining mTOR, cytokine, adhesion molecules, WNT, NFKB and NOTCH pathways were utilized to determine alterations in key markers ie: IL-12 IFN-γ, IL-4, and IL-10, IL-1, IL-4, and TNF-α etc.RESULTS:
Recipients receiving the LGR6+ ESC seeded graft when compared to skin graft controls had significantly lower levels of inflammatory markers and augmented levels of freedom from rejection. Additionally, LGR6+ ESC chimeric flaps were able to develop nascent hair follicles, which the skin controls were not capable of producing.
We hope that these preliminary findings can provide initial insight to the potential therapeutic benefit for the development of chimeric free flap allografts via the utilization of the LGR6+ ESC for tolerance induction, graft survival and hair growth . From this basic data it appears that epithelial chimerism appears to down-regulate the induction of chronic rejection.
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