Purpose

To construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation.

Methods

A stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, real-time quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks.

Results

The hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch, Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype.

Conclusion

The tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.

中文翻译：

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组织工程化的角膜上皮来源于临床级别的人类胚胎干细胞。

目的

从临床级别的人类胚胎干细胞（hESCs）构建组织工程化的角膜上皮，并研究分化过程中的动态基因图谱和表型过渡。

方法

应用了分步方案以诱导临床级hESC Q-CTS-hESC-1的分化并构建组织工程化的角膜上皮。进行单细胞RNA测序（scRNA-seq）分析以监测不同分化阶段的基因表达和表型变化。进行了免疫染色，实时定量PCR和蛋白质印迹分析以检测基因和蛋白质表达。在皮下移植到裸鼠中以测试其生物安全性之后，将上皮构建体移植到兔角膜缘干细胞缺乏症（LSCD）模型中，并随访八周。

结果

将hESC成功诱导到上皮细胞中。scRNA-seq分析揭示了分化过程中眼表上皮细胞谱系相关基因（如TP63，Pax6，KRT14）的上调以及Wnt，Notch，Hippo和Hedgehog信号通路的激活。源自hESCs的组织工程化上皮细胞片显示分层结构和正常的角膜上皮表型，并存在克隆形成的祖细胞。将细胞片移植到LSCD兔模型的眼表八周后，形成了全厚度连续角膜上皮，以完全覆盖正常角膜缘和角膜上皮表型的受损区域。

结论

由临床级hESC产生的组织工程化角膜上皮在治疗角膜缘干细胞缺乏症中可能是可行的。