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Ricardo F Frausto, Gary S L Peh, Ben L George, Jodhbir Mehta, Anthony J Aldave; Transcriptomic analysis of human corneal endothelial cells during in vitro expansion. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3795.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the effects of culturing conditions and passaging on the transcriptomic profiles of human corneal endothelial cells during in vitro expansion.
Primary human corneal endothelial cell (pHCEnC) cultures were prepared from age-matched donor corneas using one of two isolation/expansion protocols (Trypsin/F99 or Collagenase/M4M5). Cells were passaged four times. Total RNA was isolated at each passage and processed for high-throughput RNA-sequencing. Sequencing reads were aligned to the hg38 human genome and were subsequently converted to reads per kilobase per million (RPKM). Principal component analysis and hierarchical clustering were used to compare the transcriptomes at each passage. In addition, the expression of ex vivo human corneal endothelial cell (evHCEnC)-specific genes was assessed at each passage for pHCEnC cultured using each isolation/expansion protocol. Differential gene expression analysis was performed for protein-coding genes with RPKM>1 and fold-change>2. Pathway analysis was performed using the Ingenuity Pathways Analysis software.
The early passages (P0 and P1), mid-passages (P2 and P3) and late passage (P4) clustered into three distinct groups by principle component analysis (PCA). The percentage of evHCEnC-specific genes expressed in pHCEnC cultures demonstrated a decreasing trend with progressive passaging using each isolation/expansion protocol, with the percentage higher at each passage with M4M5 compared with F99. The number of differentially expressed genes (DEG) demonstrated an increasing number with progressive passaging, with a lower number at each passage (except P4) observed for the M4M5 medium. Pathway analysis of the DEG demonstrated an increase in the activation of pathways associated with cell senescence, such as IL6 and IL8. In addition, transcription of potent negative regulators of cell cycle progression, p21CIP1 and p16INK4, increased with increasing passage number.
A worldwide shortage of donor corneas has made it necessary to develop viable techniques for the expansion of human corneal endothelial cells in vitro. While the M4M5 expansion medium was associated with a higher percentage of evHCEnC-specific genes expressed and a lower number of DEG, both isolation/expansion protocols were associated with an increasing number of differentially expressed genes, and neither inhibited cell senescence.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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