Exploring the Mesenchymal Stem Cell Secretome for Corneal Endothelial Proliferation.

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.

Corneal Endothelial Cells Over the Past Decade: Are We Missing the Mark(er)?

Corneal endothelial dysfunction is one of the leading causes of corneal edema and visual impairment, requiring corneal endothelial transplantation. The treatments are limited, however, by both logistics and a global donor shortage. As a result, corneal researchers are striving to develop tissue-engineered constructs as an alternative. Recently, the clinical results of the first patients treated using a novel corneal endothelial cell therapy were reported, and it is likely many more will follow shortly. As we move from lab to clinic, it is crucial that we establish accurate and robust methods of proving the cellular identity of these products, both in genotype and phenotype. In this review, we summarized all of the markers and techniques that have been reported during the development of corneal endothelial cell therapies over the past decade. The results show the most frequently used markers were very general, namely Na+/K+ ATPase and zonula occludens-1 (ZO-1). While these markers are expressed in nearly every epithelial cell, it is the hexagonal morphology that points to cells being corneal endothelium in nature. Only 11% of articles aimed at discovering novel markers, while 30% were already developing cell therapies. Finally, we discuss the potential of functional testing of cell products to demonstrate potency in parallel with identity markers. With this review, we would like to highlight that, while this is an exciting era in corneal endothelial cell therapies, there is still no accepted consensus on a unique endothelial marker panel. We must ask the question of whether or not we are getting ahead of ourselves and whether we need to refocus on basic science rather than enter clinics prematurely.

Corneal regeneration: A review of stromal replacements.

Corneal blindness is traditionally treated by transplantation of a donor cornea, or in severe cases by implantation of an artificial cornea or keratoprosthesis. Due to severe donor shortages and the risks of complications that come with artificial corneas, tissue engineering in ophthalmology has become more focused on regenerative strategies using biocompatible materials either with or without cells. The stroma makes up the bulk of the corneal thickness and mainly consists of a tightly interwoven network of collagen type I, making it notoriously difficult to recreate in a laboratory setting. Despite the challenges that come with corneal stromal tissue engineering, there has recently been enormous progress in this field. A large number of research groups are working towards developing the ideal biomimetic, cytocompatible and transplantable stromal replacement. Here we provide an overview of the approaches directed towards tissue engineering the corneal stroma, from classical collagen gels, films and sponges to less traditional components such as silk, fish scales, gelatin and polymers. The perfect stromal replacement has yet to be identified and future research should be directed at combined approaches, in order to not only host native stromal cells but also restore functionality. STATEMENT OF SIGNIFICANCE: In the field of tissue engineering and regenerative medicine in ophthalmology the focus has shifted towards a common goal: to restore the corneal stroma and thereby provide a new treatment option for patients who are currently blind due to corneal opacification. Currently the waiting lists for corneal transplantation include more than 10 million patients, due to severe donor shortages. Alternatives to the transplantation of a donor cornea include the use of artificial cornea, but these are by no means biomimetic and therefore do not provide good outcomes. In recent years a lot of work has gone into the development of tissue engineered scaffolds and other biomaterials suitable to replace the native stromal tissue. Looking at all the different approaches separately is a daunting task and up until now there was no review article in which every approach is discussed. This review does include all approaches, from classical tissue engineering with collagen to the use of various alternative biomaterials and even fish scales. Therefore, this review can serve as a reference work for those starting in the field and but also to stimulate collaborative efforts in the future.

Xeno-Free Cultivation of Mesenchymal Stem Cells From the Corneal Stroma.

Purpose: The human cornea has recently been described as a source of corneal stroma-derived mesenchymal stem cells (hMSCs). In vitro expansion of these cells involves basal medium supplemented with fetal bovine serum (FBS). As animal-derived serum can confer a risk of disease transmission and can be subject to considerable lot-to-lot variability, it does not comply with newer Good Manufacturing Practice (GMP)-required animal component-free culture protocols for clinical translation. Methods: This study investigated animal-free alternatives to FBS for cultivation of human corneal stromal MSCs. Proliferative capacity was studied for cultures supplemented with different concentrations (2.5%, 5%, and 10%) of FBS, human AB serum, human platelet lysate (HPL), and XerumFree. Unsupplemented basal medium was used as a control. The expression of specific hMSC markers (CD73+, CD90+, CD105+, CD19-, CD34-, CD79α-, CD11b-, CD14-, CD45-, and HLA-DR-) and trilineage differentiation (adipogenesis, osteogenesis, and chondrogenesis) were compared for the two outperforming supplements: 10% FBS and HPL. Results: HPL is the only consistent non-xeno supplement where hMSC cultures show significantly higher proliferation than the 10% FBS-supplemented cultures. Both FBS- and HPL-supplemented hMSC cultures showed plastic adherence and trilineage differentiation, and no significant differences were found in the expression of the hMSC marker panel. No significant differences in stemness were detected between FBS and HPL cultures. Conclusions: We conclude that HPL is the best supplement for expansion of human corneal stromal MSCs. HPL significantly outperforms human AB serum, the chemically defined XerumFree, and even the gold standard, FBS. The xeno-free nature of HPL additionally confers preferred standing for use in GMP-regulated clinical trials using human corneal stromal MSCs.