Ex vivo grown human corneal endothelial cells (HCEnC) are a new emerging treatment option to treat visually impaired patients aimed at alleviating the current global donor shortage. Expanding HCEnC is still challenging, and obtaining cells in sufficient quantities is a limiting factor. It is already known that conditioned medium obtained from bone marrow mesenchymal stem cells can stimulate the proliferation of endothelial cells. The aim of this study was to take this work a step further to identify some of the underlying factors responsible. We confirmed the stimulatory effect of the mesenchymal stem cell secretome seen previously and separated the exosomes from the soluble proteins using size exclusion chromatography. We demonstrated the presence of exosomes and soluble proteins in the early and late fractions, respectively, with transmission electron microscopy and protein assays. Proliferation studies demonstrated that growth stimulation could be reproduced with the later protein-rich fractions but not with the exosome-rich fraction. Antibody assays revealed the presence of the secreted proteins EGF, IGFBP2, and IGFBP6 in protein-high fractions, but the growth enhancement was not seen with purified protein formulations. In conclusion, we confirmed the stimulatory effect of stem cell-conditioned medium and have determined that the effect was attributable to the proteins rather than to the exosomes. We were not able to reproduce the growth stimulation, however, with the pure recombinant protein candidates tested. Specific identification of the underlying proteins using proteomics could render a bioactive protein that can be used for ex vivo expansion of cells or as an in vivo drug to treat early corneal endothelial damage.

中文翻译：

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探索间充质干细胞分泌物组用于角膜内皮细胞增殖。

离体生长的人角膜内皮细胞（HCEnC）是一种新型的新兴治疗方法，旨在治疗视力障碍的患者，以缓解当前全球供体短缺的情况。扩展HCEnC仍具有挑战性，获得足够数量的细胞是一个限制因素。众所周知，从骨髓间充质干细胞获得的条件培养基可以刺激内皮细胞的增殖。这项研究的目的是使这项工作更进一步，以找出一些负责任的潜在因素。我们证实了先前所见的间充质干细胞分泌组的刺激作用，并使用尺寸排阻色谱法将外泌体与可溶性蛋白分离。我们证明了外泌体和可溶性蛋白分别在早期和晚期存在，用透射电子显微镜和蛋白质测定。增殖研究表明，生长刺激可以在后来的富含蛋白质的部分中复制，而不能与富含外泌体的部分一起复制。抗体检测显示高蛋白级分中存在分泌蛋白EGF，IGFBP2和IGFBP6，但纯化蛋白配方未见生长增强。总之，我们确认了干细胞条件培养基的刺激作用，并确定该作用归因于蛋白质而不是外来体。但是，通过测试的纯重组蛋白候选物，我们无法重现生长刺激。使用蛋白质组学对潜在蛋白质的特异性鉴定可以提供一种生物活性蛋白质，可用于细胞的体外扩增或作为体内药物治疗早期角膜内皮损伤。