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探索牙髓干细胞用于角膜内皮细胞生成的潜力:概念验证

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

作者信息

Bosch Begoña M, Salero Enrique, Núñez-Toldrà Raquel, Sabater Alfonso L, Gil F J, Perez Roman A

机构信息

Bioengineering Institute of Technology, Universitat Internacional de Catalunya, Barcelona, Spain.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, United States.

出版信息

Front Bioeng Biotechnol. 2021 Jan 28;9:617724. doi: 10.3389/fbioe.2021.617724. eCollection 2021.

Abstract

Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct 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.

摘要

角膜内皮细胞单层的衰竭是导致角膜移植的主要原因。需要基于自体细胞的疗法来重建细胞单层。已经提出了几种使用胚胎干细胞和诱导多能干细胞的策略,尽管它们的使用存在伦理问题以及临床应用有限。为此,我们建议使用从第三磨牙分离的牙髓干细胞来形成角膜内皮细胞单层。我们假设,使用与角膜内皮细胞具有胚胎学起源的牙髓干细胞,因为它们都起源于神经嵴,可能允许直接分化过程,避免使用重编程技术,如诱导多能干细胞。在这项工作中,我们报告了一种两步分化方案,其中牙髓干细胞先分化为神经嵴样干细胞,然后再分化为角膜内皮样细胞。最初,在第一步中,我们使用贴壁培养并比较了两种初始细胞来源:直接从牙髓干细胞形成以及从诱导多能干细胞分化。结果显示,与诱导多能干细胞相比,牙髓干细胞的早期标志物AP2水平显著更高。为了为神经嵴干细胞的生成提供更好的环境,我们采用了悬浮法,该方法诱导形成神经球。结果显示,神经球形成在4天后获得了神经嵴干细胞标志物表达的峰值,显示出AP2、巢蛋白和p75标志物的过表达,证实了神经嵴样干细胞的形成。此外,多能性标志物Oct4、Nanog和Sox2在悬浮培养中也上调。然后将神经球直接培养在角膜内皮条件培养基中进行第二次分化,成为角膜内皮样细胞。结果显示牙髓干细胞转化为表达更高水平的紧密连接蛋白1(ZO-1)、ATP酶1A1亚基(ATP1A1)、胶原蛋白4A2(COL4A2)和胶原蛋白8A2(COL8A2)标志物的多边形样细胞,证明了其转化为角膜内皮样细胞。因此,我们的研究结果表明,患者来源的牙髓干细胞可能代表一种用于角膜内皮治疗的自体细胞来源,避免了实际移植的局限性以及重编程技术。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9fa7/7876244/a5b8d069e1ec/fbioe-09-617724-g0001.jpg

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