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Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept

Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although t...

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Autores principales: Bosch, Begoña M., Salero, Enrique, Núñez-Toldrà, Raquel, Sabater, Alfonso L., Gil, F. J., Perez, Roman A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876244/
https://www.ncbi.nlm.nih.gov/pubmed/33585434
http://dx.doi.org/10.3389/fbioe.2021.617724
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author Bosch, Begoña M.
Salero, Enrique
Núñez-Toldrà, Raquel
Sabater, Alfonso L.
Gil, F. J.
Perez, Roman A.
author_facet Bosch, Begoña M.
Salero, Enrique
Núñez-Toldrà, Raquel
Sabater, Alfonso L.
Gil, F. J.
Perez, Roman A.
author_sort Bosch, Begoña M.
collection PubMed
description Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques.
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spelling pubmed-78762442021-02-12 Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept Bosch, Begoña M. Salero, Enrique Núñez-Toldrà, Raquel Sabater, Alfonso L. Gil, F. J. Perez, Roman A. Front Bioeng Biotechnol Bioengineering and Biotechnology Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques. Frontiers Media S.A. 2021-01-28 /pmc/articles/PMC7876244/ /pubmed/33585434 http://dx.doi.org/10.3389/fbioe.2021.617724 Text en Copyright © 2021 Bosch, Salero, Núñez-Toldrà, Sabater, Gil and Perez. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Bosch, Begoña M.
Salero, Enrique
Núñez-Toldrà, Raquel
Sabater, Alfonso L.
Gil, F. J.
Perez, Roman A.
Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept
title Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept
title_full Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept
title_fullStr Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept
title_full_unstemmed Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept
title_short Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept
title_sort discovering the potential of dental pulp stem cells for corneal endothelial cell production: a proof of concept
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876244/
https://www.ncbi.nlm.nih.gov/pubmed/33585434
http://dx.doi.org/10.3389/fbioe.2021.617724
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