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

ABSTRACT: Purpose: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-?? 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.

SUBMITTER: Hamuro J

PROVIDER: S-EPMC7324440 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Hamuro Junji J Numa Kohsaku K Fujita Tomoko T Toda Munetoyo M Ueda Koji K Tokuda Yuichi Y Mukai Atushi A Nakano Masakazu M Ueno Morio M Kinoshita Shigeru S Sotozono Chie C

Investigative ophthalmology & visual science 20200201 2

<h4>Purpose</h4>Aiming to clarify the metabolic interrogation in cell fate decision of cultured human corneal endothelial cells (cHCECs).<h4>Methods</h4>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 ...[more]

PMID: 32049346

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Project description:Corneal transplantation is currently the only effective treatment option for dysfunctional corneal endothelial cells (CEC). In this study, we test in vitro the surgical potential of cultivated human corneal endothelial cells (hCEC) on human anterior lens capsule (HALC), LinkCell™ bioengineered collagen sheets of 20-µm thickness (LK20), and denuded Descemet membrane (dDM) as tissue-engineered grafts for Descemet membrane (DM) endothelial keratoplasty (DMEK) to bypass the problem of donor tissue availability. Primary hCEC cultured on all carriers formed a monolayer of tightly packed cells with a high cell viability rate (96% ± 4%). hCEC on HALC and LK20 showed unremarkable expression of zonula occludens-1 (ZO-1) and Na+/K+-adenosine triphosphatase (ATPase), while Na+/K+-ATPase expression of cells seeded on dDM was mainly cytoplasmic. All hCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using a human donor cornea without DM mounted on an artificial anterior chamber (AC) and a regular DMEK-graft used as a surgical reference model. During in vitro surgery, hCEC-HALC constructs behaved most similarly to a DMEK-graft during implantation and unfolding, showing good adhesion to the bare stroma. On the other hand, hCEC-LK20 and hCEC-dDM constructs required some additional handling because of challenges related to the surgical procedure, although they were both successfully unfolded and implanted in the artificial AC. The hCEC-dDM constructs showed similar graft adherence as hCEC-HALC constructs, while adherence of hCEC-LK20 constructs was less effective. After the in vitro surgery, the estimated area populated by viable cells on the hCEC-HALC and hCEC-LK20 constructs was ∼83% and ∼67%, respectively. Overall, hCEC-HALC constructs behaved most similarly to a DMEK-graft during in vitro DMEK surgery, while graft adhesion and surgical handling, respectively, are parameters still requiring optimization for hCEC-LK20 and hCEC-dDM constructs.

Dataset Information

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

Publications

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

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