37 Differentiation of Merkel Cells Precedes Innervation in Engineered Skin Substitutes after Grafting

Abstract Introduction Skin substitutes have been shown to provide stable closure of excised burns, but relatively little is known about their innervation after grafting. Previous studies showed that Merkel cells—specialized neuroendocrine cells required for fine touch sensation—are regenerated in engineered skin after grafting. The current study investigated the timing of appearance and innervation of Merkel cells in engineered skin after transplantation using a mouse model. Methods Engineered skin was prepared using primary fibroblasts and keratinocytes isolated from de-identified human skin obtained with IRB approval. After a 10-day in vitro culture period, grafts were transplanted to full-thickness excisional wounds in immunodeficient mice (N=24). Two mice were euthanized at 2, 4, 6, 8, 10, and 12 weeks after grafting, and remaining mice were euthanized at 14 weeks. Immunohistochemistry (IHC) was performed to localize the following proteins: Merkel cell markers keratin 20 (KRT20) and keratin 18 (KRT18); neuronal markers PGP9.5, expressed in all nerves and neuroendocrine cells, and neurofilament heavy polypeptide (NFH), which is highly expressed in myelinated mature nerves; neuroendocrine marker synaptophysin (SYN); and epithelial marker N-cadherin (CDH1). Engraftment was confirmed by IHC with anti-human leukocyte antigen (HLA) antibody. Native human skin biopsies were used as positive controls. Results KRT20+ Merkel cells were not observed in engineered skin grafts prior to transplantation. At 2 weeks after grafting, small numbers of individual KRT20/KRT18-positive Merkel cells were present in the basal epidermis of engineered skin grafts but neurons were not observed. By 4 weeks, small Merkel cell clusters were observed; these cells also expressed SYN, and were rarely associated with PGP9.5-positive neurons. Merkel cell numbers appeared to increase over time and then stabilize by 10–14 weeks in vivo. Association of NFH-positive neurons with Merkel cells was observed at 8 weeks after grafting and at later time points. Merkel cells in engineered skin were confirmed to be of human epithelial origin as shown by positive staining with human-specific anti-CDH1 antibody and anti-HLA antibody. Conclusions The presence of Merkel cells in engineered skin grafts in vivo precedes innervation, suggesting that neurons are not required for Merkel cell differentiation or proliferation. The absence of Merkel cells in engineered skin in vitro and their increase in numbe