Metabolic Plasticity in Cell State Homeostasis and Differentiation of Cultured Human Corneal Endothelial Cells

Hamuro, Junji; Ueno, Masaki; Asada, Kazuko; Toda, Munetoyo; Montoya, Monty; Sotozono, Chie; Kinoshita, Shigeru

doi:10.1167/iovs.16-19807

Cited by 25 publications

(33 citation statements)

References 48 publications

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“…[2][3][4][5][6][7][8] Recently, cHCECs have been recognized to be heterogeneous not only in their biochemical features but also in their key functions, including the metabolic plasticity. 9 Corneal endothelium is one of the most metabolically active tissues in the human body. 10 Dissecting the biologic functions of metabolites and mitochondria in cHCEC cell fate decision will provide essential cues to further improve cell-based therapy.…”

mentioning

confidence: 99%

“…20 Previously, we preliminary described 20 that metabolomic profiling segregated mature differentiated-cHCECs from immature-cHCECs. 9 Cultured HCECs have a tendency for cell-state transition (CST) into a dedifferentiation state, a senescent phenotype, epithelialmesenchymal transition, and transformed fibroblastic cell morphology, indicating the presence of vast heterogeneity among these cHCECs with CST. Among cHCECs with CST, immature cHCECs switched to a glycolytic metabotype, whereas mature differentiated cHCECs became more oxidative and elicited reduced amount secretion of lactate, suggesting a possible application of metabolomics for quality control or their usefulness as biomarkers for diagnosis, prognosis, and therapeutic efficacy of cHCEC cell therapy for CE disorders.…”

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confidence: 99%

“…The secreted metabolites were assigned to the cHCEC subpopulations (SPs) distinct in the expression levels of surface CD44 antigens, 9 which expression is regulated by c-Myc. 21 The link between bioenergetics and the cell fate decision of cHCECs remains largely unknown.…”

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

Hamuro

Numa

Fujita

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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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.

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

Hamuro

Numa

Fujita

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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“…Citrate/lactate ratios discriminated CD44 À effector cells not only from CD44 þþþ , but also from CD44 þþ SPs with a minuscule amount of CST. 30 Several miRs, including miR-378a-5p, have also been shown to be involved in senescence targeting the p53 pathway. Senescence induction could provide miR-378a-5p with an additional mechanism in regard to how it is involved in CST regulation in cHCECs.…”

Section: Discussionmentioning

confidence: 99%

MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells

Ueno

Asada

Toda

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. To elucidate a noninvasive method to qualify and identify cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition and adaptable for cell-based therapy.METHODS. The variations of cHCECs in their composition of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers and their morphology. The profiles of microRNA (miRNA) in cultured cells or supernatants were detected by 3D-Gene Human microRNA Chips (Toray Industries, Inc.). The profiles were also analyzed for fresh corneal tissues with distinct endothelial cell densities (ECD) with or without gutatta. To validate the 3D-Gene results, quantitative real-time polymerase chain reaction (PCR) was performed. RNAs were extracted from cHCECs transfected with selected miRNA, and target genes were presumed by PCR array (Qiagen).RESULTS. Among a variety of morphologically different cHCECs, miRNA expression profiles were distinctively revealed. The one miRNA capable of discriminating CD44 À SP from SPs with CD44 þþ~C D44 þþþ phenotypes was identified as miR34a. The downregulation of miRNAs in the 378 family paralleled the upregulation of surface CD44 on cHCECs. Interestingly, upregulated miRNAs in the 378 family in corneal endothelium dramatically decreased in the tissues with lower ECD with advanced gutatta, providing new insight on the pathogenesis of Fuchs' endothelial corneal dystrophy. CONCLUSIONS. The specified cultured SPs sharing the CD44À surface phenotypes with matured HCECs showed the highest expression of miR-378. Conversely, SPs with upregulated CD44 þþþ showed a reduction of miR-378. Thus, miRNA in cultured cells may serve as an alternative method to qualify cHCECs.Keywords: heterogeneity of cultured corneal endothelial cells, CD44, miR, gutatta, epithelium-mesenchymal transition (EMT) A lthough human corneal endothelial cells (HCECs) have the ability to proliferate in vitro, 1 culturing HCECs for an extended period of time is known to be extremely difficult.2 To date, most researchers have conceptualized cultured HCECs (cHCECs) only from the aspect that they are derived from corneal endothelium tissue, and disregarded details pertaining to the refinement of the biochemical features. In fact, cHCECs are known to be heterogeneous from culture to culture in their morphology and in their surface markers, such as cluster-ofdifferentiation (CD) antigens.3-5 Studies, either directly or indirectly, and including those from Joyce et al., [6][7][8][9][10][11][12][13] have shown that heterogeneity is present in HCEC cultures. Of particular and striking interest is the finding by Miyai et al.3 of the presence of frequent chromosomal aneuploidy in cHCECs, directly indicating the presence of heterogeneous subpopulations (SPs) with or without aneuploidy.Cultured HCECs have an inclination toward cell-state transition (CST) into a senescence phenotype, epitheliummesenchymal transition (EMT), and fibroblastic cell morphology. In addition, the plasticity of metabolic profiles of cH...

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“…While Fuji [138] used the uniqueness of CE-MS to identify metabolites in brain tissue specific to Schizophrenia. The remainder of Table 3 lists publications related to lifestyle; alcohol intake [125], physical activity [150]], microbiome [142,145,147] and to various diseases; eye disease [140,141], inborn errors of metabolism [143], kidney disease [144], diabetes [139], metabolic syndrome [149], osteoporosis [129] and encephalopathy [148]. These publications represent a variety of subjects and sample types [serum plasma, cultured cells, PBMCs, tissue] that are amendable to CE-MS metabolomics profiling, specifically, polar hydrophilic metabolites found in critical pathways such as amino acid metabolism, central energy metabolism, inflammation, stress oxidative pathways, glycolysis, cancer metabolism, and lipid transportation.…”

Section: Medical Applicationsmentioning

confidence: 99%

Capillary electrophoresis mass spectrometry based metabolomics

Buko¹

2017

J Appl Bioanal

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IntroductionSince its first formal definition more than a decade ago, metabolomics, or, the comprehensive analysis of all metabolites present within a biological system, has attracted growing interest in clinical research by academia, industry and government labs. This is most prevalent in biomarker and drug development applications where a considerable change has been witnessed in how new diagnoses, prognoses, and therapeutic options are being discovered and developed using omic technologies. Moreover, many chronic diseases suggest a strong metabolic involvement or even a clear metabolic cause, including cancer. Together with the other omic disciplines, including genomics and proteomics, metabolomics plays a key role in the implementation of personalized medicine; evidence-based medicine designed for individually designed healthcare strategies. In turn, biomarker discovery and the understanding of biochemical pathways typically rely on a multimodal approach. Among these modalities, there continues to be a growing interest in CE-MS based development and implementation in clinical development. Formulating the problemDepending on how you classify the metabolome, there is a complex chemical space separated by hydrophilicity, polarity and size, excluding a broad range of metabolites classified as lipids, separately referred to as lipidomics. This class of metabolites also covers a large range of physical properties for which specifically designed platforms work the best. For example, for years liquid chromatography-mass spectrometry (LC-MS) has been used to capture a host of hydrophilic and hydrophobic metabolites, while gas chromatography-mass spectrometry (GC-MS) has been used to capture small molecular weight metabolites. For this review, the metabolome refers to endogenous molecules with a molecular weight typically less than 1000 Kd. To measure, catalogue, and compare the entirety of the metabolic space, the implementation of comprehensive mass spectral databases was needed (e.g., Human Metab- Because of this diversity in physical properties and size of the metabolome in biological systems, there has been a need for the development of several analytical protocols based on different chromatographic methods to select specific subgroups of metabolites based on these different chemical properties beyond the technical capabilities of LC-MS and GC-MS. These new methods have their own advantages for selecting specific types of molecules: lipids, nucleotides, aminoacids or steroids. Nonmass spectrometric methods such as nuclear magnetic resonance (NMR) and non-chromatographic methods such as matrix-assisted laser desorption-time of flight (MALDI-TOF) imaging are successfully being used for selected metabolomic analyses. The focus of this review is recent publications that use CE-MS to extend the polar metabolome beyond what is observed by LC-MS and GC-MS. Metabolomics -current methods

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Metabolic Plasticity in Cell State Homeostasis and Differentiation of Cultured Human Corneal Endothelial Cells

Cited by 25 publications

References 48 publications

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

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

MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells

Capillary electrophoresis mass spectrometry based metabolomics

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