Metabolites Interrogation in Cell Fate Decision of Cultured Human Corneal Endothelial Cells

Abstract

Aiming to clarify the metabolic interrogation in cell fate decision of cultured human corneal endothelial cells (cHCECs). METHODS. To analyze the metabolites in the culture supernatants (CS), 34 metabolome measurements were carried out for mature differentiated and a variety of cHCECs with cell state transition through a facility service. Integrated proteomics research for cell lysates by liquid chromatography−tandem mass spectrometry (LC-MS/MS) was performed for 3 aliquots of each high-quality or low-quality cHCEC subpopulations (SP). The investigations for the focused genes involved in cHCEC metabolism were performed by using DAVID and its options "KEGG_PATHWAY." RESULTS. The clusters of metabolites coincided well with the distinct content of CD44−/+ SPs. Both secreted pyruvic acid and lactic acid in the CS were negatively correlated with the content of high-quality SPs. Lactic acid and pyruvic acid in the CS exhibited the positive correlation with that of Ile, Leu, and Ser, whereas the negative correlation was with glutamine. Platelet-derived growth factor-ββ in the CS negatively correlated with lactic acid in CS, indicating indirectly the positive correlation with the content of CD44−/+ SPs. Upregulated glycolytic enzymes and influx of glutamine to the tricarboxylic acid cycle may be linked with a metabolic rewiring converting oxidative metabolism in mature differentiated CD44−/+SPs into a glycolytic flux-dependent state in immature SPs with cell state transition. CONCLUSIONS. The findings suggest that the cell fate decision of cHCECs may be dictated at least partly through metabolic rewiring.

Polarized Expression of Ion Channels and Solute Carrier Family Transporters on Heterogeneous Cultured Human Corneal Endothelial Cells

“…In a recent study, 15 our findings confirmed that the metabolic rewiring in the mature cHCECs provides essential cues underlying the dynamics of cell-fate modulation; however, and to the best of our knowledge, there have been no published reports with regard to a link between the metabolic rewiring of cHCECs and pHi homeostasis. The polarized expression of ion channels and SLC family transporters on two types of cHCECs may possibly indicate that the mature- and CST cHCECs can be distinguishable by their pHi.…”

“…CE is one of the most metabolically active tissues in the human body. 14 In our recent study, 15 our findings revealed that the metabolic rewiring in mature differentiated cHCECs provides essential cues underlying the dynamics of cell fate modulation. In the previous preliminary studies, both Wang et al 16 and our group 4 found that metabolomic profiling segregated immature from mature differentiated cHCECs.…”

“…40 We found that CD44 ++/+++ CST cHCECs display segregated profiles of extracellularly secreted metabolites (i.e., an increased amount of lactic acid and TCA-cycle intermediates), whereas secreted serine and branched-chain amino acids were decreased in the mature cHCECs. 15 …”

“…All of the following procedures were performed as recently described. 15 In brief, the cell lysates of cHCECs at passage 4 were used for the proteome analysis. Three aliquots of each lysate from the three wells for either the mature cHCECs or CST cHCECs were analyzed.…”

“…The liquid chromatography (LC)/MS dataset, composed of 4641 proteins in total, was obtained using Proteome Discoverer 2.2 software. 15 After removal of the data for which the abundance ratio could not be calculated, we analyzed the remaining data by means of the web-based program DAVID v6.8 (The Database for Annotation, Visualization and Integrated Discovery; https: //david.ncifcrf.gov). The data analysis resulted in 4315 genes, each with a unique DAVID Gene ID, for the subsequent analyses.…”