title: In vitro epithelial differentiation of human induced pluripotent stem cells for vocal fold tissue engineering.
abstract: OBJECTIVE
  We determined the feasibility and optimization of differentiating human induced pluripotent stem cells (hiPS) into nonkeratinized stratified squamous epithelial cells for vocal fold engineering.
  
  
  METHODS
  hiPS were cultured and assessed for differentiation in 3 conditions: a 3-dimensional (3D) hyaluronic acid (HA) hydrogel scaffold, a 3D HA hydrogel scaffold with epidermal growth factor (EGF), and a 3D HA hydrogel scaffold cocultured with human vocal fold fibroblasts (hVFF). After 1, 2, and 4 weeks of cultivation, hiPS were selected for histology, immunohistochemistry, and/or transcript expression analysis.
  
  
  RESULTS
  At 4 weeks, hiPS cultivated with hVFF or with EGF had significantly decreased levels of Oct 3/4, indicating loss of pluripotency. Immunofluorescence revealed the presence of pancytokeratin and of cytokeratin (CK) 13 and 14 epithelial-associated proteins at 4 weeks after cultivation in hiPS EGF and hiPS hVFF cultures. The transcript expression level of CK14 was significantly increased for hiPS hVFF cultures only and was measured concomitantly with cell morphology that was clearly cohesive and displayed a degree of nuclear polarity suggestive of epithelial differentiation.
  
  
  CONCLUSIONS
  We found that hiPS cultivated in 3D HA hydrogel with hVFF demonstrated the most robust conversion evidence to date of epithelial differentiation. Further work is necessary to focus on amplification of these progenitors for application in vocal fold regenerative biology.
authors.affiliation: Division of Otolaryngology-Head and Neck Surgery, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Imaizumi
authors.affiliation: Division of Otolaryngology-Head and Neck Surgery, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: David T
authors.initials: DT
authors.lastname: Yang
authors.affiliation: Division of Otolaryngology-Head and Neck Surgery, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Thibeault
mesh_terms: D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D008297:Male; D023822:Tissue Engineering / Q000379:methods*; D014827:Vocal Cords / Q000166:cytology*; D055815:Young Adult