Mitochondria as a Platform for Dictating the Cell Fate of Cultured Human Corneal Endothelial Cells

Abstract

Purpose Aiming to clarify the role of mitochondria in cell fate decision of cultured human corneal endothelial cell (cHCEC) subpopulations. Methods The mitochondrial respiratory ability were examined with Mito stress and Mito fuel flex test assays using an extracellular flux analyzer (XFe24; Agilent Technologies; Santa Clara, CA) for human corneal endothelium tissues, mature cHCECs and a variety of cell state transitioned cHCECs. Tricarboxylic acid cycle and acetyl-coenzyme A–related enzymes was analyzed by proteomics for cell lysates using liquid chromatography–tandem mass spectrometry for cHCEC subpopulations. Results The maximum oxygen consumption rate was found to become stable depending on the maturation of cHCECs. In the Mito stress tests, culture supplements, epidermal growth factor, SB203580, and SB431543 significantly repressed oxygen consumption rate, whereas a Rho-associated protein kinase inhibitor Y-27632 increased. Tricarboxylic acid cycle and mitochondria acetyl-coenzyme A–related enzymes were selectively upregulated in mature cHCECs, but not in cell state transitioned cHCECs. The maximum oxygen consumption rate was found to be higher in healthy human corneal endothelium tissues than those with deeply reduced cell density. An upregulated tricarboxylic acid cycle was linked with metabolic rewiring converting cHCECs to acquire the mitochondria-dependent oxidative phenotype. Conclusions Mitochondrial metabolic intermediates and energy metabolism are tightly linked to the endothelial cell fate and function. These findings will help us to standardize a protocol for endothelial cell injection.

Intracellular pH affects mitochondrial homeostasis in cultured human corneal endothelial cells prepared for cell injection therapy

“…3 a). The kinetics corresponded closely to the alteration of the increased ratios of the differentiated CEC SPs and the mitochondrial OXPHOS that we reported recently 10 . The pHi of differentiated SPs expressing the stable CD44 -/+ phenotypes on day 35 or day 42 was almost in the neutral range and was lower than that of CD44 ++/+++ CST SPs on day 35 (Fig.…”

“…In particular, mitochondrial oxidative phosphorylation (OXPHOS) was dominant in the clinically efficacious CECs. In contrast, the non-efficacious CECs in a cell-state transition (CST) 9 , 10 and characterized de-differentiation, epithelial–mesenchymal transition (EMT), immaturity, and senescence 11 , 12 showed elevated glycolysis and OXPHOS. However, it is still unclear why specified differentiated CEC SPs with OXPHOS dominance would elicit a superior improvement of corneal transparency.…”

Superiority of Mature Differentiated Cultured Human Corneal Endothelial Cell Injection Therapy for Corneal Endothelial Failure

“…For this purpose, we chose to create 'high density and mature cultured hCECs', as we intend to develop in vitro CECs that mimic in vivo CECs including energy-dependent metabolism using abundant mitochondria. 18,33 LIMITATIONS AND FUTURE CHALLENGES: It should be noted that this study did have some limitations. First, the number of eyes enrolled was small.…”

“…In fact, there is evidence from in vitro basic research supporting these phenomena. For example, the findings that mature cultured hCECs attach more to the laminin-or collagen-coated membrane than other immature CECs, 22 that they utilize a more mitochondria-dependent metabolic pathway than the glycolysis pathway, 17,18 and as revealed via proteome analysis, that they are characterized by polarized expression of several ion channels and solute carrier family transporters such as SLC4A11, NHE1, etc. 23 For comparison of the two groups at the mid-term postoperative period, contactspecular-microscopy images obtained at 3-years postoperative confirmed a higher CECD at the central cornea in the Gr2 eyes (range, 2,182-4,417 cells/mm 2 ) than in the Gr1 eyes (range, 746-2,104 cells/mm 2 ).…”

“…16 Thus, we termed the proportion of the CD24 -, CD26 -, CD44 -/dull , CD105 -/dull , CD133 -, and CD166 + cells as the "E-ratio" (see Supplemental Figure S1), and we theorize that an ideal cultured hCEC product used for cell-injection therapy should closely mimic in vivo healthy CECs (an E-ratio of nearly 100%), as they exhibit higher mitochondriadependent oxidative phosphorylation prone energy metabolism than other cultured hCEC SPs. 17,18 The cultured hCECs in Group 1 (Gr1) used for the initial study contained 0.1 to 76.3% (E-ratio) of these cells with several other types of SP cells. However, it is vital that the quality and purity of the cultured hCECs used for injection therapy is strictly controlled in regard to the homogeneity and equivalency of their composition of SPs.…”