Serum-Induced Keratinization Processes in an Immortalized Human Meibomian Gland Epithelial Cell Line

Abstract

PurposeThe aim of this study was to evaluate a human meibomian gland epithelial cell line (HMGEC) as a model for meibomian gland (patho)physiology in vitro.MethodsHMGEC were cultured in the absence or presence of serum. Sudan III lipid staining, ultrastructural analysis and lipidomic analyses were performed. Impedance sensing, desmoplakin 1/2 mRNA and cytokeratin (CK) 1, 5, 6, 14 levels were evaluated. Serum containing medium supplemented with higher serum, glucose, an omega-3 lipid cocktail, eicosapentaenoic acid or sebomed medium were investigated for lipid accumulation and ultrastructural morphology.ResultsLipid droplet accumulation in HMGEC was induced by serum containing media after 1 day, but decreased over time. Cultivation in serum induced desmosome and cytokeratin filament formation. Desmoplakin 1/2 gene levels were significantly upregulated after 1d of serum treatment. Furthermore, the normalized impedance increased significantly. Lipidome analysis revealed high levels of phospholipids (over 50%), but very low levels of wax ester and cholesteryl esters (under 1%). Stimulation with eicosapentaenoic acid increased lipid accumulation after one day.ConclusionSerum treatment of HMGEC caused lipid droplet formation to some extent but also induced keratinization. The cells did not produce typical meibum lipids under these growth conditions. HMGEC are well suited to study (hyper)keratinization processes of meibomian gland epithelial cells in vitro.

Differentiation Patterns of Immortalized Human Meibomian Gland Epithelial Cells in Three-Dimensional Culture

“…One day after medium exchange was selected based on previous investigations with 2D-cultured HMGECs. 15 In that study, lipid production in HMGECs reached a maximum 1 day after switching to differentiation medium and gradually declined thereafter. Histologic examination revealed that 1-day culture with differentiation medium promoted the apical FIGURE 2.…”

“…This finding intrigued us as recently it was shown that maturing meibocytes are connected by desmosomes and HMGECs increase cell contacts when treated with serum. 15,33 Apparently, the basally located HMGECs in direct contact with the membranes were unable to develop strong contact points comparable to desmosomes or hemidesmosomes. Interestingly, in sebocytes, extracellular matrix precoating was not necessary to form a stable multilayer.…”

“…This is also in line with previous findings demonstrating that HMGECs can differentiate to meibomian gland excretory duct lining cells. 15 CK14 is generally found in the basal cell layer of stratified epithelium 39 as well as in acini and ductal epithelium of human meibomian glands. 29 Aside from some cytoprotective functions of CK14, very little is known about CK14-related functional mechanisms, since the homozygous CK14 knockout mice show postnatal lethality of 100%.…”

“…Hampel et al 15 demonstrated that serum-induced differentiation leads to keratinization. In long-term serum treatment, CK1 and CK6 expression showed a time-dependent increase on the standard 2D format.…”

“…14 Previous studies, including some from our group, showed the functional influence of cultivation conditions, 15,16 neurotransmitters, 17,18 growth factors, 19,20 x-3 fatty acids, 21 sex hormones, 22,23 and pharmacologic substances 24 on HMGEC. However, all of these studies rely on 2D cell culture conditions or limited animal experiments.…”