April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Distinct energy metabolism between cultured mature human corneal endothelial cells and their phenotype transitioned cells
Author Affiliations & Notes
  • Kazuko Asada
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
  • Munetoyo Toda
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
  • Morio Ueno
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
  • Michiko Ujihara
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
  • Atsushi Mukai
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
  • Michio Hagiya
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
  • Naoki Okumura
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
    Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Noriko Koizumi
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
    Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  • Junji Hamuro
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Department of Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan
  • Footnotes
    Commercial Relationships Kazuko Asada, None; Munetoyo Toda, None; Morio Ueno, Santen Pharmaceutical Co (P), Senju Pharmaceutical Co (P); Michiko Ujihara, None; Atsushi Mukai, None; Michio Hagiya, JCR Pharmaceuticals Co (P); Naoki Okumura, Doshisha University (P), Pharmaceuticals Co (P), Senju Pharmaceutical Co (P); Noriko Koizumi, Doshisha University (P), JCR Pharmaceuticals Co (P), Senju Pharmaceutical Co (P); Junji Hamuro, None; Shigeru Kinoshita, Otsuka Pharmaceutical Co (C), Santen Pharmaceutical Co (P), Senju Pharmaceutical Co (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2052. doi:
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      Kazuko Asada, Munetoyo Toda, Morio Ueno, Michiko Ujihara, Atsushi Mukai, Michio Hagiya, Naoki Okumura, Noriko Koizumi, Junji Hamuro, Shigeru Kinoshita; Distinct energy metabolism between cultured mature human corneal endothelial cells and their phenotype transitioned cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2052.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The culture of cobble-stone-shape mature human corneal endothelial cells (HCECs) is damped under in vitro culture conditions by phenotypic transition; i.e., epithelial-mesenchymal transition (EMT), cell senescence, and fibrosis. To detail the molecular mechanisms underlying this phenotypic transition of HCECs in terms of the energy metabolism, we investigated the propensity of heterogeneous metabolic requirements in cultured HCECs.

Methods: To elucidate whether cultured HCECs contain subpopulations with distinct metabolic requirements, HCECs were evaluated as to their content of mitochondria (by staining with MitoTracker® Red), glycolysis, glucose uptake, and glutaminolysis. Several energy-metabolism-related functional markers were detailed in the expression of C-myc, P53, CD44, as well as senescence marker SA-β-Gal and EMT markers including α-SMA. Cultured HCEC subpopulations were sorted by flow cytometry, magnetic-activated cell sorting, or nutrient starvation, and then analyzed.

Results: Our findings showed that the failure of the long-term maintenance of cobble-stone-shape mature HCECs was partly due to the presence of the functionally heterogeneous cultured HCECs, tending to enter into phase transition to either SA-β-Gal-positive senescent state or c-Myc-positive proliferating cells (stem like) through EMT, yet the latter showed no expression of pluripotency related genes SOX2, Nanog, or Oct3/4. Compared to mature cobble-stone-shape HCECs, other phase transitioned cells required the glycolytic system for energy metabolism; i.e., mature HCECs were enriched under culture with medium starved for nutrients such as glucose. Sorted cells showed a higher frequency of mature fully differentiated HCECs defined by flow cytometry on the basis of CD166, endoglin, LGR5, and 4 other CD antigens. These results suggest that each subpopulation has distinct metabolic turnover rates and energetic requirements.

Conclusions: Our results suggest the existence of subpopulations in cultured HCECs with distinct energy metabolisms, and provide the possibility of establishing an effective method to culture HCECs containing enriched subpopulations with mature cobble-stone shapes.

Keywords: 481 cornea: endothelium • 592 metabolism  
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