Project Summary/Abstract To treat wounds that won’t heal for patients with Recessive Dystrophic Epidermolysis Bullosa, we have developed induced pluripotent cell (iPS)-cell derived cGMP compatible, scaleable induced skin composite (iSC) differentiation system that requires multi-lineage interaction to generate graftable surface ectoderm (SE), mesoderm/dermis and melanocytes. Work from ARO73170, now in its 5th year, endeavors to fill gaps in knowledge about the chromatin dynamics and interactions between the lineages to enhance manufacturing efficacy and reduce line-to-line variability. ARO73170 identified transcription factors (TFs) TFAP2 and GRHL2 that pattern early SE differentiation. In addition GATA3 and the product of the Xia-Gibbs Syndrome locus Gibbin induce Gibbin-dependent mesoderm (GDM) that matures the SE stratification program through cross-regulatory signaling. For SE differentiation, GRHL2 activity is required for TFAP2A chromatin binding and in turn, TFAP2A restricts inappropriate GRHL2 binding to ectodermal disease-associated loci, but the mechanism of interaction, and how network signaling modifies its output, remains a gap in knowledge. For GDM differentiation, we found that wild type GDM can rescue mutant ectoderm, reinforcing the need to identity the cross-regulatory stratification factors that enhance the graftability of manufactured tissue. While GATA3 binding sites are known, a gap in knowledge exists how Gibbin accumulates on key developmentally regulated promoters to activate them. Overall our preliminary data supports the intriguing hypothesis that TFAP2-GRHL2 ectoderm signaling, with reciprocal GATA3-Gibbin-GDM induction and signaling, allows proper iPS- derived graftable tissue manufacturing. We will focus on key gaps in knowledge as we dissect spatial and temporal TFAP2/GRHL2-mediated surface ectodermal commitment by dissecting the ectoderm- mesoderm lineage commitment trajectory, refining spatial constraints to optimize surface ectoderm/mesoderm commitment, interrogating GRHL2-TFAP2 mutual regulatory interactions, and modulating the GRHL2-TFAP2 target gene network to improve ectoderm commitment; and by elucidating GDM functions during skin differentiation through elucidating how Gibbin localizes to promoters of key developmental regulators, dissecting Gibbin-Dependent mesoderm signaling during mouse ectodermal development, and validating GDM-dependent factors during iPS-derived skin manufacturing. Successful completion of this proposal will provide deep mechanistic insights into the chromatin dynamics of multi-lineage tissue differentiation, highlight candidate in-process biomarkers for clinical manufacturing of skin, and establish a flexible manufacturing platform for novel cell therapy for a previously untreatable diseases.