Abstract
Purpose :
Cultured human corneal endothelial cells (cHCECs) are composed of subpopulations (SPs) that are heterogeneous in their differentiation/ maturation status. Mitochondria are responsible for coordinating cellular energy production in the vast majority of somatic cells. The purpose of this study was to clarify whether cHCEC SPs exhibit a distinctive energy metabolism in our aim to elucidate the optimal method to correctly sort cHCECs suitable for use in regenerative medicine.
Methods :
Metabolic extracts from cHCECs or corresponding culture supernatants of cHCECs were prepared and analyzed using a capillary electrophoresis (CE)-connected CE-MS/MS system (CARCINOSCOPE; HMT Inc.). Concentrations of metabolites were calculated by normalizing the peak area of each metabolite with respect to the area of the internal standard and by using standard curves. cHCECs were examined to detail the energy-metabolism-related functional markers c-Myc, CD24, CD26, and CD44.
Results :
Our findings revealed that cHCECs were composed of diverse SPs in regard to the expression of CD44, CD24, CD26, and c-Myc. Hierarchical clustering of metabolomic profiles of the culture media identified subsets of metabolites that correlated with the presence of cell-state transition (CST). SPs with CST exhibited a disposition toward anaerobic glycolysis, instead of mitochondria-dependent oxidative phosphorylation (OXHOS). Among four cHCEC SPs (i.e., C21~24), C23 showed a strong disposition toward OXHOS instead of anaerobic glycolysis, as verified by the lowest lactate production, the lowest lactate/pyruvate ratio, and the highest production of TCA cycle intermediates such as citrate/isocitrate and cis-aconitate. Interestingly, glutamine consumption was highest in C24, yet lowest in C21, indicating a difference in glutaminolysis. In our culture system, the presence of Rock-associated protein kinase (ROCK)-inhibitor Y27632 strongly induced the differentiation of cHCECs to a matured state disposed toward OXHOS with reduced CD44 expression, yet a linkage of the regulated glucose transporter type 1 translocation to the RhoA/ROCK pathway in cHCECs might be elusive
Conclusions :
Our results reveal the existence of SPs in cHCECs with distinctive energy metabolisms, and provide the possibility of establishing effective culture conditions to selectively expand mature differentiated cHCECs.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.