Human induced pluripotent stem cell-derived vocal fold mucosa mimics development and responses to smoke exposure

Abstract

Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. Here we report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa. Furthermore, we demonstrate mucosal inflammation upon exposure of these constructs to 5% cigarette smoke extract. Upregulation of pro-inflammatory genes in epithelium and fibroblasts leads to aberrant VF mucosa remodeling. Collectively, our results demonstrate that hiPSC-derived VF mucosa is a versatile tool for future investigation of genetic and molecular mechanisms underlying epithelium-fibroblasts interactions in health and disease.

RNA sequencing identifies transcriptional changes in the rabbit larynx in response to low humidity challenge

“…Although there was no gross in ammation observed, some level of epithelial cellular response to surface dehydration is expected. The vocal folds are covered by a non-keratinized strati ed squamous epithelium for which some aspects of development are well understood, such as embryological developmental factors and differentially expressed structural components (90,91), but a comprehensive molecular description is not available as for other epithelia like the epidermis. It is interesting that several keratinocyte developmental factors were identi ed with RNA-Seq and as a protein interaction cluster in the STRING analysis: CDSN, CNFN, CRNN, KRT80, KRTDAP, and TGM3.…”

Electronic cigarette vaping triggers lipid mediated vocal fold mucosal injury

“…To investigate the effect of ECVE on the human VF mucosa homeostasis, we utilized the recently developed hiPSC-derived model of human VF mucosa (15). HiPS cells were first differentiated into VF basal epithelial progenitors for 10 days and then reseeded on the top of collagen-fibroblast constructs and were let to differentiate for additional 22 days.…”

“…We have previously demonstrated that exposure of engineered VF mucosae to 5% cigarette smoke extract (CSE) leads to VF mucosa remodeling affecting predominantly the basal epithelial cell layer with downregulation of cytokeratin (K) 14 which pathologically accumulates in the luminal cells along with K13 (15). Therefore, we sought to determine whether 1-week exposure to 5% ECVE could also affect cytokeratin production and structure of the basal cellular compartment.…”

“…In this section, we will primarily focus on the mucin and cytokine/chemokine expression. We evaluated expression of Mucin1 (MUC1) and 4 (MUC4) which are typical membrane-associated mucins present in the human VF (12,15). Secretory proteins, such as MUC5B or MUC5C, are not produced by stratified VF epithelial cells that cover the membranous portion of the true VF (12).…”

“…The recent development of a three-dimensional (3D) model of human VF mucosa by our group has allowed us to mimic in vivo remodeling of the VF mucosa in tobaccorelated diseases (15). Upon exposure to 5% cigarette smoke extract (CSE) for 1 week, we were able to induce keratotic changes and mucosal inflammation in engineered VF mucosae, composed of human induced pluripotent stem cell (hiPSC) derived VF epithelial cells and primary human VF fibroblasts dispersed in the collagen gel that mimicked the lamina propria.…”