Skip to main content
Article
Human Embryonic Stem Cell-Derived Epithelial Cells in a Novel In Vitro Model of Vocal Mucosa
Tissue Engineering Part A (2013)
  • Ciara Leydon, Sacred Heart University
  • Joshua A. Selekman
  • Sean Palecek
  • Susan L. Thibeault
Abstract

Introduction: Tissue engineering represents a promising approach for esophageal replacement, considering the complexity and drawbacks of conventional techniques.

Aim: To create the components necessary to reconstruct in vitro or in vivo an esophageal wall, we analyzed the feasibility and the optimal conditions of human and pig skeletal myoblast (HSM and PSM) and porcine oral epithelial cell (OEC) culture on biologic scaffolds.

Materials and Methods: PSM and HSM were isolated from striated muscle and porcine OECs were extracted from oral mucosa biopsies. Myoblasts were seeded on an acellular scaffold issue from porcine small intestinal submucosa (SIS) and OEC on decellularized human amniotic membrane (HAM). Seeding conditions (cell concentrations [0.5×106 versus 106 cells/cm2] and culture periods [7, 14 and 21 days]), were analyzed using the methyl thiazoltetrazolium assay, quantitative PCR, flow cytometry, and immunohistochemistry.

Results: Phenotypic stability was observed after cellular expansion for PSM and HSM (85% and 97% CD56-positive cells, respectively), and OECs (90% AE1/AE3- positive cells). After PSM and HSM seeding, quantities of viable cells were similar whatever the initial cell concentration used and remained stable at all time points. During cell culture on SIS, a decrease of CD56-positive cells was observed (76% and 76% by D7, 56% and 70% by D14, 28% and 60% by D21, for PSM and HSM, respectively). Multilayered surface of α-actin smooth muscle and Desmine-positive cells organized in bundles was seen as soon as D7, with no evidence of cell within the SIS. Myoblasts fusion was observed at D21. Pax3 and Pax7 expression was downregulated and MyoD expression upregulated, at D14.OEC proliferation was observed on HAM with both cell concentrations from D7 to D21. The cell metabolism activity was more important on matrix seeded by 106 cells/cm2. With 0.5×106 OEC/cm2, a single layer of pancytokeratin-positive cells was seen at D7, which became pluristratified by D14, while when 106 OEC/cm2 were used, a pluristratified epithelial structure was seen as soon as D7. Proliferative cells (Proliferating Cell Nuclear Antigen staining) were mainly located at the basal layer.

Conclusion: In this model, the optimal conditions of cell seeding in terms of cell concentration and culture duration were 0.5×106 myoblasts/cm2 and 106 OEC/cm2, and 7 days.

Keywords
  • esophageal replacement,
  • in vitro,
  • in vivo,
  • methyl thiazoltetrazolium assay,
  • quantitative PCR,
  • flow cytometry,
  • immunohistochemistry,
  • Phenotypic stability,
  • cell seeding,
  • culture duration
Publication Date
October, 2013
Publisher Statement
Basic Business Terms Authorized Users are defined as employees, faculty, staff and students who are officially affiliated with the Licensee, and persons affiliated with remote sites or campuses of the Licensee that are administered from the Licensee's site or campus (but not persons affiliated with remote sites or campuses with a separate administrative staff). Authorized Users may also include users from the general public who are permitted by the Subscribing Institution to access the Journals from designated terminals within the Subscribing Institution's IP site. Fees are due and payable by Licensee thirty (30) days after the date of invoice from Licensor. All amounts payable by the Licensee under this Agreement shall be exclusive of any sales, use, value added or similar taxes. Scope of License Agreement This License Agreement permits simultaneous access to the Licensed Material by the Licensee's Authorized Users. Remote access by authorized users via the subscribing institution's proxy server is permitted. Access to the available archive of the Licensed Material will be provided at no additional charge with a current, paid subscription. Post-Cancellation Access terms, outlined in 6 below, will apply to this archive material. Responsibility The Licensee shall take reasonable steps to prevent unauthorized use. Licensee will not be liable to the Publisher for breach of the terms of this agreement so long as licensee did not intentionally assist in or encourage such breach or permit such breach to continue after having actual notice thereof. The Licensee will make reasonable efforts to ensure that there is no unauthorized access, and to correct in a reasonable time any violations that have been brought to the Licensee's attention by written notification from the Publisher. Permitted Use The Authorized Users are permitted online access to the Licensed Material and may download, save, or print text, search results or other information solely for the private use or research of the Authorized Users. The Licensee may use the Journals for educational resources such as "electronic reserves" and "class-packs" provided solely to Authorized Users, subject to normal reproduction fees under copyright law. The electronic form may be used as a source for Inter-Library Loan in compliance with Section 108 of the United States Copyright Law (17 USC §108, "Limitations on exclusive rights: Reproduction by libraries and archives") and the Guidelines for the Proviso of Subsection 108(2g)(2) prepared by the National Commission on New Technological Uses of Copyrighted Works. Prohibitions on Certain Uses Licensee may not remove, obscure or modify any copyright or other notices included in the Licensed Material. Altering, recompiling, systematic or programmatic copying, reselling, redistributing, publishing or re-publishing the Journal content or any part thereof without explicit permission from the copyright holder is prohibited. Systematic downloading, (e.g., spidering), service bureau redistribution services, printing for "fee-for-service" purposes and/or the systematic making of electronic copies for transmission to non-subscribers are prohibited. Downloading of Journals or parts of Journals to form a persistent local archive is prohibited. Withdrawal of Journals or Parts of Journals The Licensor reserves the right to withdraw from this Agreement any of the Journals or any part of any of the Journals in order to comply with any legal rule or court order, or if the Licensor is at risk of liability from the continued availability of the Journal or part of it. Post-Cancellation Access Post-Cancellation Access rights are granted to the volume year of the Licensed Material to which a subscription was paid. The Licensee may acquire Post-Cancellation Access rights to the archive, volume years of the Licensed Material, for which no subscription fee has been paid, on payment of a one-time fee. This fee will be based on the number of articles from the Licensed Material included in the archive. After the date of expiry or termination of this Agreement the Licensee will, subject to the terms and conditions set forth in this Agreement, continue to have access to the Licensed Material for which Post-Cancellation Rights have been paid. Such access will be at no additional charge providing the Licensee maintains at least one, current electronic subscription to content published by the Licensor. In the event that the Licensee fails to maintain a current electronic subscription to content published by the Licensor, the Licensor will continue to provide access to the Licensed Material only upon payment of an annual access fee. Copyright Copyright in the Licensor's Journals and their contents, including the abstracts, is vested in the Licensor, whose proprietary and publishing rights are protected under national and international law. Neither Authorized Users nor the Licensee shall have any proprietary right in any of the Journals by reason of their access to them. Usage Statistics The Licensor warrants that its platform gives librarians/administrators functionality to generate COUNTER-compliant statistics. Licensor's undertakings The Licensor shall indemnify and hold the Licensee harmless from and against any direct loss, damage, cost, liability or expense (including reasonable legal and professional fees) arising out of any legal action taken against the Licensee claiming that the Licensee's use of the Licensed Material within the terms permitted in this License Agreement infringes any copyright or other proprietary or intellectual property rights of any natural or legal person. This indemnity shall survive the termination of this License Agreement for any reason. This indemnity shall not apply if the Licensee has modified the Licensed Material in any way not permitted by this License Agreement. The Licensor shall: Make the Licensed Material available to the Licensee and to Authorized Users from the Licensor's server. Make available to the Licensee the electronic version of each journal covered by this License Agreement, no later than reasonably promptly after publication of the printed version. Provide the Licensee with information sufficient to enable access to the Licensed Material. The Licensor shall use all reasonable endeavors to: Make the Licensed Materials available to the Licensee and to Authorized Users at all times and on a twenty-four hour basis, save for routine maintenance Ensure that its server has adequate capacity and bandwidth to support the usage of the Licensee at a level commensurate with the standards of availability for information services of similar scope operating via the World Wide Web, as such standards evolve from time to time over the term of this License Agreement. Provide the Licensee with adequate and competent technical support and assistance to enable the Licensee to use the Licensed Material. The Licensor will not disclose to any third party any information about the Licensee's business, including without limitation the list of Licensed Material or any usage statistics relating to particular journals or particular articles, without the prior written consent of the Licensee. If you have any questions about the Mary Ann Liebert, Inc., Licensing Agreements please do not hesitate to contact our Sales Department at sales@liebertpub.com
Citation Information
Ciara Leydon, Joshua A. Selekman, Sean Palecek and Susan L. Thibeault. "Human Embryonic Stem Cell-Derived Epithelial Cells in a Novel In Vitro Model of Vocal Mucosa" Tissue Engineering Part A Vol. 12 Iss. 19-20 (2013)
Available at: http://works.bepress.com/ciara_leydon/4/