Mitsuyoshi Imaizumi1, Yuka Sato1, David T Yang2, Susan L Thibeault1. 1. Division of Otolaryngology-Head and Neck Surgery, University of Wisconsin-Madison, Madison, Wisconsin, USA. 2. Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Abstract
OBJECTIVES: 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.
OBJECTIVES: 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: Dinesh K Chhetri; Christian Head; Elena Revazova; Stephen Hart; Sunita Bhuta; Gerald S Berke Journal: Otolaryngol Head Neck Surg Date: 2004-12 Impact factor: 3.497
Authors: Xiao Zheng Shu; Kaustabh Ghosh; Yanchun Liu; Fabio S Palumbo; Yi Luo; Richard A Clark; Glenn D Prestwich Journal: J Biomed Mater Res A Date: 2004-02-01 Impact factor: 4.396