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INTRODUCTION

Corneal endothelial transplantation accounts for most of corneal transplantation for treating corneal diseases, however severe shortage of corneal donors is the biggest obstacle. In our previous study, we differentiated human skin-derived precursors (SKPs) into corneal endothelial cell (CEC)-like cells with a co-culture system. In this study, we aimed to investigate cell differentiation molecular mechanism and evaluate the function of CEC-like cells by developing tissue-engineered corneas in order to improve cell production efficiency and provide basic research for clinical transformation.

METHODS

We performed transcriptome sequencing of SKPs and CEC-like cells. Further, we focused on the possible enriching pathways, including PI3K/Akt, MAPK/Erk, WNT/β-catenin, and important transcription factors Pitx2 and Foxc1. The PI3K and β-catenin inhibitors were also added to the culture system to observe the differentiation alteration. We developed a graft for a tissue-engineered cornea (TEC) using CEC-like cells and acellular porcine cornea matrix scaffold. The tissue-engineered corneas were transplanted into rabbits via penetrating keratoplasty.

RESULTS

The PI3K/Akt, MAPK/Erk, and WNT/β-catenin pathways play important roles during the differentiation of SKPs into CEC-like cells. Crosstalk existed between the PI3K/Akt and MAPK/Erk pathways. The PI3K/Akt and WNT/β-catenin pathways were connected. Pitx2 and Foxc1 were subject to temporal and spatial controls of the WNT/β-catenin pathway. The inhibition of the PI3K/Akt and WNT/β-catenin pathways both prevented cell differentiation. CEC-like cells grew well on the acellular porcine cornea matrix scaffold, and the tissue-engineered corneal graft performed well after transplantation into rabbits.

CONCLUSION

We provide experimental basis for CEC-like cell industrial production and drive the cells to be clinically applied in cellular replacement therapy or alternative graft substitution for treating corneal diseases in the future.

INTRODUCTION

METHODS

RESULTS

CONCLUSION

引言

角膜内皮移植是治疗角膜疾病的主要角膜移植方式，但角膜供体严重短缺是最大障碍。在我们之前的研究中，我们通过共培养系统将人皮肤来源的前体细胞（SKP）分化为角膜内皮细胞（CEC）样细胞。在本研究中，我们旨在通过构建组织工程角膜来研究细胞分化的分子机制并评估CEC样细胞的功能，以提高细胞生产效率并为临床转化提供基础研究。

方法

我们对SKP和CEC样细胞进行了转录组测序。此外，我们关注了可能的富集通路，包括PI3K/Akt、MAPK/Erk、WNT/β-连环蛋白以及重要转录因子Pitx2和Foxc1。还向培养系统中添加了PI3K和β-连环蛋白抑制剂以观察分化变化。我们使用CEC样细胞和脱细胞猪角膜基质支架构建了组织工程角膜移植物。通过穿透性角膜移植术将组织工程角膜移植到兔子体内。

结果

PI3K/Akt、MAPK/Erk和WNT/β-连环蛋白通路在SKP分化为CEC样细胞的过程中起重要作用。PI3K/Akt和MAPK/Erk通路之间存在串扰。PI3K/Akt和WNT/β-连环蛋白通路相互关联。Pitx2和Foxc1受WNT/β-连环蛋白通路的时空调控。PI3K/Akt和WNT/β-连环蛋白通路的抑制均阻止了细胞分化。CEC样细胞在脱细胞猪角膜基质支架上生长良好，组织工程角膜移植物移植到兔子体内后表现良好。