Human Embryonic Stem Cell-Derived Epithelial Cells in a Novel In Vitro Model of Vocal Mucosa

A satisfactory in vitro model of vocal fold mucosa does not exist, thus precluding a systematic, controlled study of vocal fold biology and biomechanics. We sought to create a valid, reproducible three-dimensional (3D) in vitro model of human origin of vocal fold mucosa of human origin. We hypothesized that coculture of human embryonic stem cell (hESC)-derived simple epithelial cells with primary vocal fold fibroblasts under appropriate conditions would elicit morphogenesis of progenitor cells into vocal fold epithelial-like cells and creation of a basement membrane. Using an in vitro prospective study design, hESCs were differentiated into cells that coexpressed the simple epithelial cell marker, keratin 18 (K18), and the transcription factor, p63. These simple epithelial cells were cocultured with primary vocal fold fibroblasts seeded in a collagen gel scaffold. The cells were cultured for 3 weeks in a keratinocyte medium at an air-liquid interface. After that time, the engineered mucosa demonstrated a stratified, squamous epithelium and a continuous basement membrane recapitulating the key morphologic and phenotypic characteristics of native vocal fold mucosa. hESC-derived epithelial cells exhibited positive staining for vocal fold stratified, squamous epithelial markers, keratin 13 (K13) and 14 (K14), as well as tight junctions, adherens junctions, gap junctions, and desmosomes. Despite the presence of components critical for epithelial structural integrity, the epithelium demonstrated greater permeability than native tissue indicating compromised functional integrity. While further work is warranted to improve functional barrier integrity, this study demonstrates that hESC-derived epithelial progenitor cells can be engineered to create a replicable 3D in vitro model of vocal fold mucosa featuring a multilayered, terminally differentiated epithelium.