Transcriptional Profiling Provides New Insights into Organ Culture-Induced Changes in Human Donor Corneas

Abstract

Corneal transplantation is one of the most common forms of tissue transplantation worldwide. Donor corneal tissue used in transplantation is provided by eye banks, which store the tissue in culture medium after procurement. To date, the effects of cell culture on human corneal tissue have not been fully elucidated. Using the 3′ RNA sequencing method for massive analysis of cDNA ends (MACE), we show that cultivation of corneal tissue leads to significant changes in a variety of molecular processes in human corneal tissue that go well beyond aspects of previously known culture effects. Functionally grouped network analysis revealed nine major groups of biological processes that were affected by corneal organ culture, among them keratinization, hypoxia, and angiogenesis, with genes from each group being affected by culture time. A cell type deconvolution analysis revealed significant modulations of the corneal immune cell profile in a time dependent manner. The results suggest that current culture conditions should be further refined and that prolonged cultivation may be detrimental. Recently, we showed that MACE enables transcriptional profiling of formalin-fixed and paraffin-embedded (FFPE) conjunctival tissue with high accuracy even after more than 10 years of storage. Here we demonstrate that MACE provides comparable results for native and FFPE corneal tissue, confirming that the technology is suitable for transcriptome analysis of a wide range of archived diseased corneal samples stored in histological archives. Finally, our data underscore the feasibility of bioinformatics cell-type enrichment analysis in bulk RNA-seq data to profile immune cell composition in fixed and archived corneal tissue samples, for which RNA-seq analysis of individual cells is often not possible.

Transcriptional profiling specifies the pathogen-specific human host response to infectious keratitis

“…Corneal tissue processing was performed as previously described ( Wolf et al., 2022b ). Briefly, after corneas were retrieved, the tissue was fixed in 4% formalin (phosphate-buffered, pH 7) for 12 hours at room temperature, dehydrated in alcohol, and processed for paraffin embedding.…”

“…Fifteen FFPE sections, each 4 µm thick, were taken from each corneal sample and stored in RNase-free tubes prior to RNA extraction, as previously described ( Wolf et al., 2022b ).…”

“…RNA extraction from corneal samples was performed by a commercial provider (GenXPro, Frankfurt am Main, Germany) as previously described ( Wolf et al., 2022b ). Briefly, total RNA was isolated from FFPE samples using the Quick-RNA FFPE Kit (Zymo Research, Irvine, CA, USA).…”

“…Previous studies demonstrated the value of RNA sequencing (RNA-seq) to examine disease mechanisms at high resolution and in an unbiased approach ( Wolf et al., 2023 ). The technology enables detailed molecular characterization of human corneal tissue ( Collin et al., 2021 ), even in formalin-fixed and paraffin-embedded (FFPE) corneal specimens, that are available from histological biobanks ( Boneva et al., 2020 ; Wolf et al., 2022b ). In particular, the cellular (especially immunological) compartments have been described ( Wolf et al., 2020 ; Zhuang et al., 2022 ) and it has been shown how the cellular composition of corneal grafts, the tissue that will later be used for corneal transplantation, is altered by tissue culture in eye bank storage ( Zhuang et al., 2022 ; Wolf et al., 2022b ).…”

“…The technology enables detailed molecular characterization of human corneal tissue ( Collin et al., 2021 ), even in formalin-fixed and paraffin-embedded (FFPE) corneal specimens, that are available from histological biobanks ( Boneva et al., 2020 ; Wolf et al., 2022b ). In particular, the cellular (especially immunological) compartments have been described ( Wolf et al., 2020 ; Zhuang et al., 2022 ) and it has been shown how the cellular composition of corneal grafts, the tissue that will later be used for corneal transplantation, is altered by tissue culture in eye bank storage ( Zhuang et al., 2022 ; Wolf et al., 2022b ). Using microarray technology, another study demonstrated gene expression changes in bacterial and fungal keratitis ( Chidambaram et al., 2017 ).…”