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Cornea  |   June 2014
Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells
Author Affiliations & Notes
  • David A. Sullivan
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Yang Liu
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Wendy R. Kam
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Juan Ding
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Karin M. Green
    Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Shrewsbury, Massachusetts, United States
  • Scott A. Shaffer
    Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Shrewsbury, Massachusetts, United States
  • Mark P. Hatton
    Ophthalmic Consultants of Boston, Boston, Massachusetts, United States
  • Shaohui Liu
    Schepens Eye Research Institute, Boston, Massachusetts, United States
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
Investigative Ophthalmology & Visual Science June 2014, Vol.55, 3866-3877. doi:10.1167/iovs.13-13407
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      David A. Sullivan, Yang Liu, Wendy R. Kam, Juan Ding, Karin M. Green, Scott A. Shaffer, Mark P. Hatton, Shaohui Liu; Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(6):3866-3877. doi: 10.1167/iovs.13-13407.

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

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Abstract

Purpose.: We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells.

Methods.: Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression.

Results.: Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells.

Conclusions.: Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland.

Introduction
The differentiation of meibomian gland epithelial cells is critically important for the health and well-being of the ocular surface. These cells produce meibum, a lipid-rich mixture that is released by the gland at the eyelid margins. Meibum spreads onto the tear film and serves to stabilize this film, prevent its evaporation, and promote visual acuity. 1,2 Interference with this lipid-related production and secretion, such as occurs in meibomian gland dysfunction (MGD), leads to tear film instability and evaporation 13 and is the major cause of dry eye disease throughout the world. 1,4  
Unfortunately, despite the importance of meibomian gland epithelial cells, almost nothing is known about the control of their differentiation. Insofar as we know today, meibomian gland epithelial cell differentiation in vivo, as with other sebaceous gland epithelial cells, begins with small, undifferentiated cells located in the acinar periphery. These cells contain large numbers of free ribosomes and mitochondria, and a poorly developed smooth endoplasmic reticulum (SER) and Golgi apparatus. As cells mature and start their migration toward the lateral ductules, the SER and Golgi become more prominent, lysosomes are generated, and lipids begin to accumulate. Ultimately, cells terminally differentiate, a process associated with a dramatic increase in volume, a profusion of lipid-filled vesicles, and nuclear pyknosis. Cells then undergo holocrine secretion, which involves cellular autophagy, apoptosis, disintegration, and release of lipid-laden contents into the ductules. 1,59  
A preclinical model of this cellular differentiation process in vitro would be ideal. Such a model not only would permit the discovery of agents that regulate this cellular maturation, but also allow the development of new pharmaceutical approaches for the treatment of MGD. 
We hypothesize that immortalized human meibomian gland epithelial cells can serve as this preclinical model in vitro. We further hypothesize that exposure of these cells to serum will induce differentiation and provide the opportunity to identify factors that control this process. The purpose of this investigation was to begin to test our hypotheses, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. 
Methods
Meibomian Gland Epithelial Cell Culture Procedures
Primary and immortalized human meibomian gland epithelial cells 10,11 were cultured in keratinocyte serum-free medium (SFM) containing 5 ng/mL epidermal growth factor (EGF) and 50 μg/mL bovine pituitary extract (BPE). After reaching 70% to 90% confluence, cells were placed in SFM or serum-containing medium (10% fetal bovine serum in equal volumes of Dulbecco's modified Eagle's medium and Ham's F12 with 10 ng/mL EGF) for varying intervals. Media and serum were purchased from Invitrogen-Gibco (Grand Island, NY, USA). The use of human tissues was approved by the Institutional Review Boards of the Schepens Eye Research Institute and Massachusetts Eye and Ear and adhered to the tenets of the Declaration of Helsinki. 
Lysosome and Neutral Lipid Staining
After designated time intervals, immortalized human meibomian gland epithelial cells were cultured for 30 minutes in the presence of LysoTracker Red DND-99 (Invitrogen-Gibco) to label lysosomes. 12 Cells then were fixed in paraformaldehyde, processed for staining with LipidTOX green neutral lipid stain (Invitrogen-Gibco) and 4′,6-diamidino-2-phenylindole (DAPI) blue nuclear stain (Invitrogen-Gibco), and mounted with ProLong Gold antifade, as described previously. 11 Cells were imaged for LysoTracker (excitation/emission, 577/590 nm) and LipidTox (excitation/emission, 495/505 nm) staining with a Nikon Eclipse E800 (Nikon Instruments, Melville, NY, USA). 
Cellular Lipid Analyses
Following the designated culture period, primary and immortalized human meibomian gland epithelial cells were treated with 0.25% trypsin and EDTA (Invitrogen-Gibco), washed with PBS twice, then centrifuged. Cell pellets were stored at −80°C until thawing for lipid analyses. Lipids were extracted by the method of Folch et al. 13  
For neutral lipid (i.e., cholesterol, cholesterol esters, wax esters) analysis, a lipid aliquot (100 μg) was further extracted with a Bond Elut NH2 Solid Phase Extraction 50 mg cartridge (Agilent Technologies, Columbia, MD, USA). In brief, the aliquot was dried, resuspended in 1:1 hexane:isopropanol and applied to the conditioned cartridge. The flow-through was collected, dried, resuspended in mobile phase A, and injected with internal standards into a LUNA C18(2) 100Å 150 × 1.00 mm HPLC column (Phenomenex, Torrance, CA, USA) using a Surveyor Autosampler and LC-pump (Thermo Fisher Scientific, Waltham, MA, USA), and analyzed in triplicate. Elution of the C18 column was performed with (A) 95% methanol, 5% 100 mM ammonium formate, pH 4.75 and (B) 95% isopropanol, 5% 100 mM ammonium formate, pH 4.75. The flow rate was set at 50 μL/min and the gradient was: 0 minutes 50% B, 5 minutes 50% B, 20 minutes 100% B, 95 minutes 100% B, 96 minutes 0% B with a 24-minute re-equilibration time at each run's end. Full scan spectra from a mass spectrometer (MS) were obtained by using a positive ion electrospray MS and a Orbitrap Velos Pro (Thermo Fisher Scientific) at 60,000 resolution. Neutral lipid standards used in these analyses included 1-heptadecanoyl-rac-glycerol (17:0 MG) > 99%; 1,2-dilauroyl-sn-glycerol (12:0/12:0 DG) > 99%; 1,3-ditetradecanoyl-2-(9Z-hexadecenoyl)-glycerol (14:0/16:1/14:0 TG) > 99%; cholest-5-en-3β-yl pentadecanoate (15:0 cholesterol ester) > 99%; and 1-oleoyl-N-heptadecanoyl-D-erythro-sphingosine (17:0/d18:1 Ceremide) > 99% (Avanti Lipids, Alabaster, AL, USA); cholesterol-D7 > 98% (Cambridge Isotope Lab, Andover, MA, USA); and oleyl laurate wax ester (Nu-Check-Prep, Inc., Elysian, MA, USA). The neutral lipids standards were mixed to 0.4 ng/μL triglycerides, 1 ng/μL diglyceride/cholesterol ester/wax ester, 2 ng/μL monoglyceride/ceremide, and 20 ng/μL cholesterol, and then diluted 1:1 with the sample extract. Statistical analysis of cholesterol levels was performed with Student's unpaired t-test. 
For polar lipid analysis, a lipid aliquot (10 μg) and an internal standard mixture were injected into a LUNA 3 μ Silica(2) 100Å 150 × 1.00 mm column (Phenomenex) using a Surveyor Autosampler and LC-pump (Thermo Fisher Scientific), and analyzed by light chromatography/mass spectrometry (LC/MS) in triplicate and LC/MS2/MS 3 in singlicate. Elution of the Silica column was carried out with (A) 58:40:02, isopropanol:hexane:100 mM ammonium formate, pH 4.75 and (B) 50:40:10, isopropanol:hexane:100 mM ammonium formate, pH 4.75. The flow rate was 50 μL/min and the gradient was: 0 minutes 0% B, 15 minutes 0% B, 40 minutes 100% B, 50 minutes 100% B, 51 minutes 0% B, with a 40-minute re-equilibration time at the end of each run. Full scan MS spectra were obtained using negative ion electrospray MS and a LTQ Orbitrap Velos (Thermo Fisher Scientific) at 60,000 resolution. The data-dependent MS 2 and MS 3 scans were acquired on the most intense fragment ion of the top 5 intense ions in the full MS spectra. This process was used to facilitate the identification of the fatty acid composition of individual polar lipid molecular species. The chromatographic elution time for each lipid group was determined by observing the elution of the internal standard and the highest molecular weight species. The extracted ion current of each lipid molecular species was processed with SIEVE software (Thermo Fisher Scientific), and the lipid identity was determined by the retention time and exact mass (5 parts per million [ppm]). Identities were verified by using the fragment ions and the extracted ion chromatogram profiles. The phospholipid standards purchased from Avanti Lipids included: dimyristoyl phosphocholine (14:0/14:0 PC) > 99%; dimyristoyl phosphoethanolamine (14:0/14:0 PE) > 99%; dimyristoyl phosphoserine (14:0/14:0 PS) > 99%; dimyristoyl phosphoglycerol (14:0/14:0 PG) > 99%; dimyristoyl phosphate (14:0/14:0 PA) > 99%; dilauroyl sphingosylphosphocholine (d18:1/12:0 SM) > 99%;1-myristoyl phosphate (14:0 LPA) > 99%; 1-heptadecenoyl phosphoserine (14:0 LPS) > 99%; 1-myristoyl phosphoethanolamine (14:0 LPE) > 99%; and 1-myristoyl phosphoglycerol (14:0 LPG) > 99%. The polar lipids standards were mixed to 0.1 ng/μL of PG, 1 ng/μL of PE, 2 ng/μL of LPG/LPE/PS/LPS/LPA/SM, and 20 ng/μL of PA/PC. 
For all lipid groups except phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, the ion detected was the molecular species minus one proton. For the excepted species, the ion detected was the molecular ion minus methane plus acetate. The average intensities of all ions were obtained from four replicate measurements. 
Microarray Procedures
Total RNA was isolated from cells, analyzed for integrity and processed for the measurement of mRNA levels at Asuragen (Austin, TX, USA), as previously published. 14 Our experiments used Illumina HumanHT-12 v3 and v4 Expression BeadChips (Illumina, San Diego, CA, USA). Data were acquired with Illumina BeadStudio software, and used background subtraction and cubic spline normalization. Standardized hybridization intensity values were adjusted by adding a constant, so that the lowest intensity value for any sample was equal to 16. 15  
Normalized data were evaluated without log transformation and statistical analyses were performed with Student's t-test (2-tailed, unpaired). These analyses were conducted with GeneSifter software (Geospiza, Seattle, WA, USA), which also yielded gene ontology, KEGG pathway, and z-score (zsc) reports organized according to the guidelines of the Gene Ontology Consortium (available in the public domain at http://www.geneontology.org/GO). 16 Gene comparisons between groups were facilitated by using the GeneSifter intersector program (Geospiza; available in the public domain at www.public.genesifter.net). All data from the Illumina BeadChips may be obtained and downloaded from the National Center for Biotechnology Information's Gene Expression Omnibus (available in the public domain at http://www.ncbi.nlm.nih.gov/geo) via series accession numbers GSE18099 and GSE 37089. 
Real-Time PCR Procedures
The differential expression of selected genes was verified by using quantitative real-time PCR (qPCR) methods. The cDNAs were generated by using SuperScript III Reverse Transcriptase (Invitrogen-Gibco) and random hexamer primers (Invitrogen-Gibco). The qPCR reactions were carried out in triplicate with TaqMan Gene Assays (Applied Biosystems, Inc., Foster City, CA, USA), TaqMan-specific primers and probes for lipocalin 2 (Hs.204238), claudin 1 (Hs.439060), acetyl-coenzyme A acetyltransferase 2 (Hs.571037), ornithine decarboxylase 1 (Hs.467701), and GAPDH (4326317E). Differential gene expression was calculated by following the Comparative Ct method, as described in Applied Biosystems User Bulletin 2 (updated 2001). 
Results
Effect of Serum on Gene Expression in Human Meibomian Gland Epithelial Cells
To determine whether serum upregulates genes associated with cellular differentiation, we cultured primary and immortalized human meibomian gland epithelial cells in SFM for 5 days (“undifferentiated” cells), another 2 days to 70% to 80% confluence, followed by an additional 14 days in either SFM or serum-containing medium. Cells then were processed for analyses with Illumina BeadChips and Geospiza software. 
Our results showed that serum clearly stimulates the activity of genes related to cell differentiation. As demonstrated in Table 1, serum exposure significantly (P < 0.05) altered the expression of over 2750 genes in primary and immortalized human meibomian gland epithelial cells. This effect was found irrespective of whether serum-associated responses were compared to those of undifferentiated or SFM-treated cells. The magnitude of serum's influence was quite remarkable. For example, serum exposure induced 19- to 456-fold increases in the activity of genes encoding retinoic acid receptor responder 1, chemokine (C-C motif) ligand 28 (CCL28), insulin-like growth factor binding protein 3, matrix metallopeptidase 12, phospholipase A2, and prominin (Table 2). Many of these genes are associated with cell differentiation. Serum also enhanced by up to 5.9-fold the gene expression of transforming growth factor β2, increased the mRNA levels of netrin 4 and collagen, type V, α2 by 2.5- to 21.1-fold, and induced the appearance of polymeric immunoglobulin receptor mRNA (pIgR; data not shown). Especially noteworthy was our finding that these molecular responses were very similar between primary and immortalized human meibomian gland epithelial cells (Tables 2 1552 1552 1552 15527). Indeed, serum elicited the same responses (i.e., significant up- or downregulation compared to “undifferentiated”) in over 2500 genes in both primary and immortalized human meibomian gland epithelial cells. 
Table 1
 
Influence of Serum on Gene Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 1
 
Influence of Serum on Gene Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Group Genes ↑ Genes ↓ Total Genes
Undiff vs. serum
 Primary 2125 2358 4483
 Immortalized 2025 2787 4812
SFM vs. serum
 Primary 1368 1814 3182
 Immortalized 1602 1187 2789
Table 2
 
Effect of Serum on Gene Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 2
 
Effect of Serum on Gene Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Accession # Gene Ratio Ontology
Upregulation
 NM_000478 Alkaline phosphatase 24.7, 11.9, 12.3, 11.9 Anatomical structure morphogenesis
 NM_019846 chemokine (C-C motif) ligand 28 15.7, 19.1, 18.9, 17.6 Chemotaxis
 NM_000598 Insulin-like growth factor binding protein 3 54.2, 33.8, 41.3, 20.8 Regulation of cell growth
 NM_002426 Matrix metallopeptidase 12 50.1, 45.6, 43.0, 54.8 Proteolysis
 NM_007069 Phospholipase A2, group XVI 55.4, 17.9, 24.3, 9.0 Lipid catabolic process
 NM_006017 Prominin 1 32.4, 31.9, 32.4, 31.9 Cell differentiation
 NM_206963 Retinoic acid receptor responder (tazarotene induced) 1 456.0, 207.0, 30.6, 99.5 Negative regulation of cell proliferation
 NM_019554 S100 calcium binding protein A4 24.1, 20.8, 12.3, 9.8 Epithelial to mesenchymal transition
 NM_003357 Secretoglobin, family 1A, member 1 31.2, 18.3, 29.3, 17.7 Negative regulation of transcription from RNA polymerase II promoter
 NM_030754 Serum amyloid A2 140.9, 32.2, 7.2, 43.6 Acute phase response
 NM_003186 Transgelin 62.0, 107.5, 8.1, 11.9 Anatomical structure development
Downregulation
 NM_001218 Carbonic anhydrase XII 11.9, 9.8, 8.8, 14.4 1-Carbon metabolic process
 NM_002775 HtrA serine peptidase 1 5.7, 6.5, 4.9, 7.5 Negative regulation of TGF β receptor signaling pathway
The influence of serum on genes associated with cell differentiation also was quite evident from zsc analyses of biological process, molecular function, and cellular component ontologies in human meibomian gland cells. Compared to undifferentiated or SFM controls, serum induced a significant increase in ontologies related to the endoplasmic reticulum, Golgi apparatus, lysosomes, vesicles, growth factor activity, cell differentiation, epithelium development, and vesicle-mediated transport (Tables 3–5). Conversely, serum also caused a significant decrease in the expression of genes linked mitochondria, ribosomes, cell cycle, mitosis and translation (Tables 3 15525). Examples of gene activities in the tissue development ontology that were significantly altered by serum are shown in Table 6
Table 3
 
Effect of Serum Exposure on Cellular Component Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 3
 
Effect of Serum Exposure on Cellular Component Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Ontology Undifferentiated vs. Serum SFM vs. Serum
Pri Genes Pri z-Score Imm Genes Imm z-Score Pri Genes Pri z-Score Imm Genes Imm z-Score
Upregulation
 Cytoplasmic membrane-bounded vesicle 97 3.14 89 2.99 68 3.2 81 4.07
 Cytoplasmic vesicle 105 3.5 101 3.94 72 3.29 89 4.64
 Endomembrane system 213 5.4 183 3.99 161 6.65 176 6.57
 Endoplasmic reticulum 144 4.14 120 2.6 105 4.69 132 6.7
 Endoplasmic reticulum membrane 90 3.41 81 3.03 71 4.69 81 5.17
 Endoplasmic reticulum part 101 3.55 86 2.51 82 5.27 95 6.02
 Golgi apparatus 162 7.25 143 6.26 107 5.9 128 7.26
 Golgi apparatus part 106 6.71 88 5.04 68 5.1 84 6.65
 Golgi cisterna 16 2.96 15 2.95 13 3.5 16 4.32
 Golgi cisterna membrane 13 2.51 11 2 10 2.73 13 3.72
 Golgi lumen 6 2.48 6 2.72 4 2.04 5 2.56
 Golgi membrane 84 5.56 73 4.65 61 5.56 72 6.53
 Golgi stack 24 4.29 20 3.41 18 4.31 21 4.87
 Lysosome 65 7.66 54 6.11 31 3.24 30 2.31
 Lytic vacuole 65 7.66 54 6.11 31 3.24 30 2.31
 Membrane-bounded vesicle 100 3.32 92 3.2 70 3.34 84 4.33
 Vacuole 69 8.26 69 7.73 38 3.88 34 2.25
 Vesicle 111 3.88 106 4.23 76 3.59 94 5.02
Downregulation
 Cytosolic large ribosomal subunit 28 12.9 22 8.67 29 15.54 15 9.42
 Cytosolic small ribosomal subunit 29 14.19 20 8.2 32 18.23 13 8.44
 Large ribosomal subunit 20 10.75 17 7.96 13 7.55 5 3
 Mitochondrial envelope 84 5.93 84 4.31 69 5.78 37 2.8
 Mitochondrial large ribosomal subunit 11 7.27 9 5.06 7 4.94 4 3.29
 Mitochondrial nucleoid 15 6.37 11 3.52 7 2.62 5 2.38
 Mitochondrial part 149 10.63 151 8.66 112 8.65 51 2.84
 Mitochondrion 265 12.63 292 12.05 202 10.51 102 4.34
 Organellar large ribosomal subunit 11 7.27 9 5.06 7 4.94 4 3.29
 Ribonucleoprotein complex 220 24.93 201 19.29 163 20.3 60 6.66
 Ribosome 113 22.93 93 16.03 98 22.68 35 8.33
Table 4
 
Influence of Serum on Biological Process Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 4
 
Influence of Serum on Biological Process Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Ontology Undifferentiated vs. Serum SFM vs. Serum
Pri Genes Pri z-Score Imm Genes Imm z-Score Pri Genes Pri z-Score Imm Genes Imm z-Score
Upregulation
 Anatomical structure development 349 2.64 334 3.35 256 4.28 291 4.4
 Apoptosis 195 6 184 6.13 132 5.21 159 6.43
 Apoptotic mitochondrial changes 11 3.68 9 2.89 10 4.69 8 2.97
 Cell adhesion 117 4.49 96 2.78 97 6.52 103 5.88
 Cell death 209 5.85 199 6.17 142 5.16 166 5.88
 Cell differentiation 237 2.25 223 2.49 162 2.38 195 3.38
 Cell migration 87 4.13 76 3.31 62 4.11 71 4.4
 Cell morphogenesis 86 2.81 75 2.07 62 3.11 73 3.64
 Cellular response to stimulus 500 4.25 446 3.07 337 3.99 387 4.31
 Epidermis development 42 4.72 46 6.23 28 3.86 27 2.82
 Epithelium development 65 4.3 61 4.28 46 4.09 49 3.67
 Induction of apoptosis 60 3.99 59 4.47 36 2.44 54 5.16
 Induction of programmed cell death 60 3.95 59 4.43 37 2.62 54 5.11
 Organ morphogenesis 76 2.24 75 2.84 53 2.29 72 4.24
 Programmed cell death 196 5.99 185 6.12 134 5.36 160 6.45
 Proteolysis 132 5.22 130 5.98 87 4.18 108 5.59
 Signal transduction 430 4.31 391 3.71 294 4.3 328 3.95
 Signaling 453 3.21 412 2.65 314 3.77 347 3.11
 Tissue development 136 5.27 128 5.33 98 5.39 111 5.61
 Vesicle-mediated transport 115 4.36 103 3.76 77 3.67 80 2.79
 Wound healing 84 3.67 71 2.55 59 3.56 75 5.01
Downregulation
 Cell cycle 209 7.95 327 15.98 124 2.54 84 2.34
 Cell cycle checkpoint 56 7.04 77 9.92 28 2.25 22 2.85
 Cell cycle phase 154 9.09 248 17.46 79 2.22 54 2.11
 Cell cycle process 168 8 275 16.69 95 2.37 64 2.12
 Cell division 79 6.01 139 13.58 47 2.47 37 3.32
 Gene expression 505 9.8 555 8.19 412 9.61 226 3.75
 M phase 92 6.38 172 15.94 52 2.12 37 2.26
 M phase of mitotic cell cycle 78 7.33 143 16.41 40 2.2 29 2.39
 Mitosis 76 7.31 139 16.26 39 2.23 29 2.59
 Mitotic cell cycle 151 10.08 233 17.53 76 2.72 50 2.17
 NcRNA metabolic process 93 12.6 96 11.27 71 10.55 32 4.49
 NcRNA processing 66 10.51 68 9.37 54 9.73 25 4.45
 Nuclear division 76 7.31 139 16.26 39 2.23 29 2.59
 Nuclear export 39 9.3 30 5.31 30 7.88 14 3.76
 Ribonucleoprotein complex assembly 33 8.13 30 6.03 19 4.44 10 2.38
 Ribonucleoprotein complex biogenesis 91 15.81 80 11.5 70 13.43 27 4.84
 Ribonucleoprotein complex subunit organization 34 8.06 31 5.97 20 4.52 11 2.63
 Ribonucleotide metabolic process 18 4.82 17 3.63 58 3.28 36 2.17
 Ribosome biogenesis 69 15.54 60 11.38 61 15.71 20 4.84
 RNA processing 172 13.95 180 12.43 126 10.82 52 3.11
 rRNA metabolic process 47 11.72 44 9.39 41 11.7 17 4.98
 rRNA processing 46 11.88 43 9.53 40 11.8 17 5.24
 Sterol biosynthetic process 19 6.39 19 5.52 9 2.52 10 4.56
 Translation 180 21.45 157 15.28 151 20.43 67 9.34
 Translational elongation 75 19.97 55 12.1 77 23.76 32 11.05
 Translational initiation 27 7.6 26 6.21 23 7.41 12 4.27
 Translational termination 68 19.2 52 12.31 74 24.37 31 11.52
Table 5
 
Impact of Serum on Molecular Function Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 5
 
Impact of Serum on Molecular Function Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Ontology Undifferentiated vs. Serum SFM vs. Serum
Pri Genes Pri z-Score Imm Genes Imm z-Score Pri Genes Pri z-Score Imm Genes Imm z-Score
Upregulation
 Cysteine-type endopeptidase activity 13 2.74 12 2.57 9 2.46 13 3.9
 Cytoskeletal protein binding 70 2.76 62 2.13 58 4.49 56 2.99
 Enzyme binding 94 2.92 93 3.49 68 3.42 72 2.75
 Growth factor activity 23 3.8 17 2.19 23 6.15 21 2.78
 Peptidase activity 78 3.38 84 4.91 54 3.29 68 4.5
Downregulation
 DNA-dependent ATPase activity 16 3.38 25 5.94 12 2.73 8 2.17
 Electron carrier activity 31 3.28 31 2.29 24 2.8 16 2.21
 GTP binding 60 3.67 64 2.92 49 3.61 29 2
 GTPase activity 44 4.76 41 2.95 29 2.81 23 3.32
 Ligase activity 64 2.46 83 3.77 57 3.36 37 2.43
 mRNA binding 25 6.13 24 4.84 21 5.96 9 2.28
 Nucleic acid binding 437 7.33 492 6.55 351 7.1 211 3.51
 Nucleotide binding 345 8.25 389 7.76 250 5.57 150 2.67
 Purine nucleotide binding 257 4.97 310 5.94 191 3.46 126 2.52
 Purine ribonucleoside triphosphate binding 253 5.06 309 6.32 187 3.44 126 2.8
 Purine ribonucleotide binding 255 4.93 309 6 189 3.38 126 2.6
 Ribonucleotide binding 255 4.93 309 6 189 3.38 126 2.6
 RNA binding 251 19.9 223 13.73 198 17.62 89 7.18
 Structural constituent of ribosome 109 24.09 86 16.09 99 25.05 38 10.23
 Structure-specific DNA binding 36 3.92 44 4.63 32 4.46 18 2.47
 Transferase activity, transferring alkyl or aryl (other than methyl) groups 12 3.17 12 2.54 13 4.65 7 2.67
 Translation factor activity, nucleic acid binding 33 8.51 32 7.05 25 7.12 12 3.46
Table 6
 
Serum-Induced Alteration of Genes Related to Tissue Development in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 6
 
Serum-Induced Alteration of Genes Related to Tissue Development in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Accession # Gene Ratio Ontology
Upregulation
 NM_000598 Insulin-like growth factor binding protein 3 54.2, 33.8, 41.3, 20.8 Regulation of cell growth
 NM_005046 Kallikrein-related peptidase 7 52.5, 80.9, 4.5, 2.9 Epidermis development
 NM_001878 Cellular retinoic acid binding protein 2 44.8, 20.9, 2.0, 3.8 Epidermis development
 NM_000900 Matrix Gla protein 39.2, 3.5, 33.2, 4.8 Protein complex assembly
 NM_001323 Cystatin E/M 31.3, 59.2, 3.2, 5.6 Epidermis development
 NM_002167 Inhibitor of DNA binding 3 21.5, 28.5, 3.3, 5.5 Negative regulation of transcription
 NM_021229 Netrin 4 21.1, 4.2, 6.3, 3.7 Neuron remodeling
 NM_172037 Retinol dehydrogenase 10 18.0, 5.2, 8.4, 3.3 Organ morphogenesis
 NM_019554 S100 calcium binding protein A4 17.5, 20.8, 8.4, 3.5 Cell differentiation
 NM_002166 Inhibitor of DNA binding 2 14.9, 36.8, 5.3, 3.1 Negative regulation of transcription
 NM_004936 Cyclin-dependent kinase inhibitor 2B 14.4, 15.9, 5.0, 3.7 Regulation of cyclin-dependent protein kinase activity
 NM_001901 Connective tissue growth factor 14.0, 9.2, 4.6, 10.5 Angiogenesis
 NM_001453 Forkhead box C1 11.4, 8.8, 3.2, 3.1 Blood vessel development
 NM_000393 Collagen, type V, α2 7.6, 3.9, 2.5, 8.8 Axon guidance
 NM_001955 Endothelin 1 6.1, 4.1, 2.0, 4.1 Prostaglandin biosynthetic process
 NM_001018004 Tropomyosin 1α 4.6, 8.4, 2.1, 6.0 Cellular component movement
 NM_000693 Aldehyde dehydrogenase 1 family, member A3 2.9, 2.5, 3.1, 3.5 Retinoic acid biosynthetic process
 NM_000362 TIMP metallopeptidase inhibitor 3 2.2, 4.6, 2.8, 2.7 Central nervous system development
 NM_001039348 EGF-containing fibulin-like extracellular matrix protein 1 2.1, 2.4, 1.9, 12.6 Epidermal growth factor receptor signaling pathway
 NM_000963 Prostaglandin-endoperoxide synthase 2 2.0, 7.2, 5.6, 13.7 Prostaglandin biosynthetic process
Downregulation
 NM_003068 Snail homolog 2 5.6, 4.2, 3.1, 2.2 Negative regulation of transcription
 NM_005213 Cystatin A 5.2, 3.2, 4.3, 16.9 Negative regulation of peptidase activity
 NM_000575 Interleukin 1α 4.5, 3.6, 2.2, 2.6 Inflammatory response
 NM_033260 Forkhead box Q1 4.2, 5.2, 2.5, 3.7 DNA fragmentation involved in apoptotic nuclear change
 NM_006142 Stratifin 4.1, 2.3, 2.4, 2.0 Regulation of cyclin-dependent protein kinase activity
 NM_000067 Carbonic anhydrase II 4.0, 2.6, 13.3, 7.3 1-Carbon metabolic process
 NM_004429 Ephrin-B1 4.0, 2.9, 2.2, 2.2 Neural crest cell migration
These differentiative effects were duplicated in another series of experiments, which involved the exposure of immortalized human meibomian gland epithelial cells (n = 3 wells/condition) to serum-containing or serum-free media for 4 days. Serum treatment significantly (P < 0.05) increased ontologies and pathways linked to cytoplasmic vesicles (zsc = 4.7), epithelium development (zsc = 3.3), Golgi apparatus (zsc = 8.4), insulin signaling pathway (zsc = 2.1), lysosomes (zsc = 7.2), vesicle-mediated transport (zsc = 7.0), and autophagy (zsc = 4.0), and decreased those associated with cell cycle (zsc = 16.2), focal adhesion (zsc = 3.6), mitosis (zsc = 15.0), mitochondrion (zsc = 10.0), ribosomes (zsc = 7.1), and translational elongation (zsc = 4.2). 
The differentiating impact of serum on primary and immortalized human meibomian gland epithelial cells also was demonstrated by the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. As shown in Table 7, serum enhanced the activity of lysosome, phagosome and p53 signaling pathways, and decreased those associated with cell cycle and ribosomes. 
Table 7
 
Effect of Serum on KEGG Pathways in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 7
 
Effect of Serum on KEGG Pathways in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Ontology Undifferentiated vs. Serum SFM vs. Serum
Pri Genes Pri z-Score Imm Genes Imm z-Score Pri Genes Pri z-Score Imm Genes Imm z-Score
Upregulation
 Lysosome 35 6.5 31 5.96 21 4.29 17 2.29
 p53 signaling pathway 18 4.15 19 5.05 12 3.26 16 4.51
 Phagosome 37 5.54 31 4.57 25 4.45 26 4.02
Downregulation
 Aminoacyl-tRNA biosynthesis 12 3.1 17 4.96 11 3.58 7 2.82
 Cell cycle 36 5.3 51 8.51 24 3.43 21 4.83
 Ribosome 69 18.22 50 11.32 74 23.08 31 11.08
 RNA transport 51 7.58 57 8.19 40 6.71 17 2.45
 Steroid biosynthesis 10 5.04 10 4.66 6 3.05 6 4.43
To verify in part the Illumina BeadChip results, selected genes were analyzed by qPCR. This experimental approach confirmed the regulatory effects of serum on lipocalin 2, claudin 1, ornithine decarboxylase 1, and acetyl-coenzyme A acetyltransferase 2 (Table 8). 
Table 8
 
Confirmation of Selected BeadChip Gene Expression Results
Table 8
 
Confirmation of Selected BeadChip Gene Expression Results
Gene Cell SFM BeadChip SFM qPCR Serum BeadChip Serum qPCR
Upregulation
 LCN2 Primary 6.2 7.5 72.0 161.9
 LCN2 Imm 22.6 34.0 21.7 40.3
 CLDN1 Primary 15.1 13.9 10.2 11.5
 CLDN1 Imm 8.1 9.0 8.6 9.8
Downregulation
 ODC1 Primary 7.8 12.1 21.9 19.7
 ODC1 Imm 4.5 5.9 6.9 14.6
 ACAT2 Primary 10.3 15.4 12.4 17.4
 ACAT2 Imm 4.3 3.4 7.1 9.5
Influence of Serum on Neutral and Polar Lipid Expression in Human Meibomian Gland Epithelial Cells
To examine whether serum-induced differentiation is paralleled by changes in the lipid profile of primary and immortalized human meibomian gland epithelial cells, we analyzed the neutral and polar lipid content in cells after the 2-week culture in SFM or serum-containing medium. 
Our results showed that serum exerts quantitative and qualitative alterations in neutral and polar lipid patterns of human meibomian gland epithelial cells. In addition, the nature of the serum-related response was similar in primary and immortalized cells. Serum exposure was associated with a significant (P < 0.05, 1-tail) 34% increase in cholesterol levels (primary [P] + immortalized [I] SFM = 189.1 ± 24.5 ng, P + I serum = 253.4 ± 39.9 ng). Similarly, serum treatment led up to 34-fold differences in the amount of various fatty acids in wax and cholesterol esters (Table 9). 
Table 9
 
Influence of Serum on Wax and Cholesterol Ester Fatty Acid Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 9
 
Influence of Serum on Wax and Cholesterol Ester Fatty Acid Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Fatty Acid Serum-Free Media Serum-Containing Media
Primary Cells Immortalized Cells Primary Cells Immortalized Cells
Wax ester
 33:1 587.0, 476.6 707.2, 897.2 166.7, 115.8 291.8, 236.1
 41:3 58.0, 41.9 24.3, 25.2 162.9, 92.4 78.7, 121.5
 42:2 56.4, 84.2 37.7, 67.2 234.5, 205.0 291.4, 292.7
Cholesterol ester
 20:3 89.0, 216.2 166.2, 176.8 854.7, 1023.9 818.1, 1216.9
 20:4* 70.4, 364.5* 220.6, 470.3* 744.7, 1152.0* 887.9, 1192.5*
 22:4 1.0, 8.5 6.8, 5.7 148.0, 173.7 154.8, 279.6
The response to serum-induced differentiation appeared to be most pronounced in cellular phospholipids. As shown in Table 10, primary and immortalized human meibomian gland epithelial cells contained many different phospholipids, including phosphatidylcholine, sphingomyelin, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, phosphatidic acid, phosphatidylglycerol, lysophosphatidylcholine, lysophosphatidylinositol, and phosphatidylinositol. Phosphatidylcholine was expressed at the highest level (i.e., 47%–57% of total measured phospholipids). Culture of cells in serum resulted in numerous alterations in the type and amount of fatty acids in most of the phospholipid species. Two very consistent changes were the often dramatically increased levels (e.g., up to 109-fold greater) of polyunsaturated fatty acids, as well as arachidonic acid, in differentiated cells (Table 11). 
Table 10
 
Phospholipid Expression in Human Primary and Immortalized Meibomian Gland Epithelial Cells In Vitro
Table 10
 
Phospholipid Expression in Human Primary and Immortalized Meibomian Gland Epithelial Cells In Vitro
Phospholipid Serum-Free Media Serum-Containing Media
Primary Cells Immortalized Cells Primary Cells Immortalized Cells
Phosphati-dylcholine 57.00, 52.35 49.43, 43.93 55.97, 50.63 47.04, 44.97
Sphingomyelin 19.93, 16.91 20.55, 20.47 13.49, 15.45 18.47, 17.95
Lysophosphati-dylethanolamine 11.24, 13.16 11.98, 15.48 14.13, 15.30 14.77, 16.89
Lysophospha-tidylserine 3.70, 5.01 5.61, 7.03 6.45, 6.76 7.24, 7.97
Phosphatidyl-serine 2.39, 4.69 4.17, 4.53 4.05, 5.27 5.47, 4.91
Phosphatidyl-ethanolamine 2.47, 3.63 3.29, 3.05 2.59, 3.60 3.02, 2.68
Lysophosphati-dylglycerol 1.55, 2.26 2.46, 2.55 1.94, 1.29 1.51, 2.67
Lysophosphatidic acid 1.27, 1.17 1.41, 1.46 0.91, 1.12 1.57, 1.22
Phosphatidic acid 0.41, 0.70 0.97, 1.31 0.41, 0.51 0.84, 0.63
Phosphati-dylglycerol 0.05, 0.13 0.13, 0.19 0.08, 0.07 0.07, 0.10
Table 11
 
Effect of Serum Exposure on Phospholipid Fatty Acid Content in Human Primary and Immortalized Meibomian Gland Epithelial Cells
Table 11
 
Effect of Serum Exposure on Phospholipid Fatty Acid Content in Human Primary and Immortalized Meibomian Gland Epithelial Cells
Phospholipid Serum-Free Media Serum-Containing Media
Primary Cells Immortalized Cells Primary Cells Immortalized Cells
Phosphatidylcholine
 38:2 2480, 2976 2798, 3794 1,795, 828.2 1,687, 1,168
 38:4* 567.2, 1206* 699.4, 647.2* 14,642, 9,083* 5,157, 5,304*
 38:5 277.4, 629.3 338.3, 292.5 10,131, 6,107 4,206, 4,535
 38:6 97.2, 156.1 66.0, 59.9 2,565, 1,674 1,169, 1,115
Lysophosphatidylethanolamine
 20:4* 2155, 5363* 2192, 3504* 19,861, 12,622* 11,603, 15,859*
 22:2 204.2, 386.6 364.0, 1091 75.6, 45.7 104.5, 77.5
 22:4 312.6, 811.0 585.6, 1457 3,154, 2,254 2,220, 3,016
 22:6 456.0, 1080 479.1, 916.8 21,845, 16,015 12,697, 14,728
Lysophosphatidylserine
 22:5 193.3, 521.9 240.6, 481.3 2,080, 1,383 1,449, 1,585
 22:6 75.8, 222.9 103.6, 198.4 3,872, 2,435 2,111, 2,756
Phosphatidylserine
 40:6 56.7, 235.0 107.5, 160.8 2,836, 2,301 1,866, 1,897
 40:7 17.8, 51.3 66.7, 25.0 221.3, 213.1 164.3, 131.4
 42:9 0.4, 1.8 0.5, 0.8 118.8, 104.9 81.9, 79.6
Phosphatidylethanolamine
 38:2 312.8, 569.5 440.2, 618.0 167.0, 142.5 203.1, 118.4
 38:4* 330.0, 975.4* 365.8, 404.6* 2,970, 2,792* 1,810, 1,753*
 38:6 30.7, 73.2 26.1, 26.9 551.7, 571.7 329.5, 321.0
 40:7 17.0, 49.9 35.3, 35.3 925.7, 898.9 685.5, 616.5
Lysophosphatidylglycerol
 20:2 212.4, 823.6 585.1, 973.0 3,608, 1,574 1,550, 3,348
 20:4* 39.2, 131.8* 119.3, 155.1* 642.5, 277.9* 232.3, 569.1*
 22:5 21.8, 85.0 24.7, 39.6 530.4, 205.6 232.2, 490.5
 22:6 19.3, 75.2 18.8, 37.2 2,261, 1,029 970.4, 2,117
Phosphatidylglycerol
 40:7 0.6, 4.8 1.7, 3.9 117.1, 53.6 51.7, 97.0
All our neutral and polar lipid raw data are shown in the Supplementary Tables
The exposure of human meibomian gland epithelial cells to serum, as we previously discovered, 11 was associated with the acquisition of lipid-containing vesicles. As demonstrated in the Figure, much of this lipid accumulation appeared to occur within lysosomes. 
Figure
 
Lipid accumulation in immortalized human meibomian gland epithelial cells. Cells were cultured in serum for 5 or 13 days, and then processed for the identification of neutral lipids with LipidTOX, lysosomes with LysoTracker Red DND-99, and nuclei with DAPI. The figures were overlaid with Adobe Photoshop (Adobe Systems, San Jose, CA, USA) to create the “Merge” figure.
Figure
 
Lipid accumulation in immortalized human meibomian gland epithelial cells. Cells were cultured in serum for 5 or 13 days, and then processed for the identification of neutral lipids with LipidTOX, lysosomes with LysoTracker Red DND-99, and nuclei with DAPI. The figures were overlaid with Adobe Photoshop (Adobe Systems, San Jose, CA, USA) to create the “Merge” figure.
Discussion
Our results supported our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells in vitro. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipid-containing organelles (e.g., lysosomes), as well as alterations in the fatty acid content of neutral and polar lipids. Of particular importance, our results showed that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are similar to those of primary cells. Overall, our findings indicated that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cell differentiation in vitro. 
Our hypothesis that exposure of human meibomian gland epithelial cells to serum would induce differentiation, compared to proliferation, was prompted by several considerations. First, serum has been shown to promote the differentiation of other epithelial cell types, including those from human foreskin, mammary, bronchial, and corneal tissues. 1823 This developmental process is associated with an upregulation of genes related to cell differentiation and tissue development, and a downregulation of genes linked to cell cycle and cell division. 24,25 Second, serum is known to reduce or inhibit the proliferation of many types of epithelial cells, including those from the trachea, bronchus, mammary gland, urothelium, conjunctiva, and oral mucosa. 1820,2628 We also recently have found that serum decreases the proliferation of primary and immortalized human meibomian gland epithelial cells, compared to that of cells cultured in SFM. 10,11 Third, we have discovered that serum treatment increases the accumulation of neutral lipids in immortalized human meibomian gland epithelial cells. 10,11 This response is characteristic of differentiation in other sebaceous gland epithelial cells, 11,29,30 as well as in adipocytes. 31,32 Of interest, we observed in the present study that lipid accumulation in differentiated meibomian gland epithelial cells appears to occur, at least in part, in lysosomes. 
The serum-induced differentiation of human meibomian gland epithelial cells is associated not only with an increased expression of tissue development genes, but also those associated with anti-inflammatory and antibacterial activities. For example, serum upregulates the gene encoding secretoglobin, family 1A, member 1 (i.e., uteroglobin), which suppresses inflammation, 33 and downregulates the gene for interleukin 1α, a proinflammatory cytokine. In addition, serum enhances the expression of genes for phospholipase A2, group XVI, and CCL28. Phospholipase A2 kills gram-positive bacteria and is a key bactericide in human tears. 34 Also, CCL28 has antimicrobial activity against gram-positive and gram-negative bacteria, and Candida albicans . 35 These transcripts, if translated, could contribute to the absence of inflammation and bacterial invasion within the meibomian gland in obstructive MGD. 14,3638  
It also is of interest that CCL28 attracts IgA-positive cells to mucosal tissues. 39 Given that serum stimulates pIgR gene expression in primary human meibomian gland epithelial cells, it is possible that these differentiated cells could have a role in transporting polymeric IgA antibodies to the ocular surface and protecting against microbial infection. In support of this concept, we previously have identified IgA α chain in human meibomian gland secretions, 40 and others have shown that sebaceous glands can secrete IgA. 41  
A particularly intriguing finding was the observation that serum upregulates the expression of genes encoding netrin 4 and collagen, type V, α2. These proteins are involved with neuron remodeling and axon guidance, and, if translated and secreted in vivo during cellular differentiation, could possibly influence meibomian gland innervation. The meibomian gland is the only sebaceous gland that is heavily innervated, 5 and human meibomian gland epithelial cells are responsive to neurotransmitters. 42 However, essentially nothing is known about the control of meibomian gland innervation in vivo. 
We discovered that the process of differentiation is associated with significant alterations in the amount of cholesterol, as well as the fatty acid profiles of wax esters, cholesterol esters and phospholipids, in both primary and immortalized human meibomian gland epithelial cells. That such a response can occur is not surprising, given that meibomian glands contain the transcripts necessary for all the key lipogenic enzymes involved in cholesterol and fatty acid synthesis. 43,44 Further, the expression of these enzymatic mRNAs is known to be significantly increased by factors (e.g., androgens) that promote differentiated functions in meibomian gland epithelial cells. 4346  
It is possible that the quantitative and qualitative changes in fatty acid levels during cellular differentiation might be influenced by the relative activity of several enzymes. These include: phospholipase A2, group XVI, which catalyzes the release of fatty acids from phosphatidylcholine and phosphatidylethanolamine 33 ; secretoglobin, family 1A, member 1, which may inhibit phospholipase A2 33 ; and prostaglandin-endoperoxide synthase 1 and 2, also known as cyclooxygenase (COX) 1 and 2, which convert free arachidonic acid after its release from membrane phospholipids by phospholipase A2 to prostaglandin H2. 33 The gene expression for these enzymes is significantly altered during serum-stimulated differentiation of human meibomian gland epithelial cells. 
Two of the most striking changes during cellular differentiation were the increases in arachidonic acid content in, and polyunsaturation of, phospholipid fatty acids. If released by the action of phospholipase A2, arachidonic acid could serve as the precursor of many biologically active products (i.e., eicosanoids) that modulate epithelial cell growth and differentiation. 47,48 Arachidonic acid also may be metabolized by 5-lipoxygenase, ultimately yielding leukotriene B4 (LTB4). 4749 The LTB4 is secreted by human meibomian gland epithelial cells in response to bacterial toxin exposure 50 and theoretically could promote ocular surface inflammation. It remains to be determined whether these changes in arachidonic acid levels also occur in vivo during differentiation. It is possible that they reflect increased arachidonic acid uptake from the serum-containing media in vitro. 5153 As concerns the increased polyunsaturation, it is of interest that a decreased unsaturation of nonpolar fatty acids has been associated with MGD. 5456  
The human meibomian gland epithelial cell lipids detected in our study do not reflect the levels of neutral and polar lipids typically found in human meibum. 2,5760 The reason is that we analyzed cellular extracts, not meibum, and the lipid components of these products are not necessarily the same. To explain, the meibomian gland secretes through a holocrine process, which involves disintegration of the whole cell and secretion of the entire cell components into the lateral ductile and, ultimately, the central and terminal ducts. 1,59 This would include the membranes, which are enriched in phospholipids. However, ductal cells, as in other exocrine glands, often alter the content of their luminal secretions. Such ductal activity also has been proposed for sebaceous glands. 61 More specifically, Thiboutot speculated that phospholipids are recycled in sebaceous gland ducts after holocrine secretion, resulting in the delivery of predominantly neutral lipids to the skin. 61 This recycling process also may occur in the meibomian gland, which would explain, for example, why meibum is depleted of phospholipids. 2,5759 Further research is required to determine the precise role of ductal cells in modulating the lipid content in the human meibomian gland. 
Lastly, our study demonstrated that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are similar to those of primary cells. This observation is particularly important, given that other immortalized human ocular surface cells do not necessarily mimic their primary cell counterparts, and that their responses may not be physiologically relevant 62,63 Overall, our results indicated that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cell differentiation. 
Supplementary Materials
Acknowledgments
The authors express their appreciation to Barbara and James E. Evans (Worcester, Massachusetts, United States) for their technical assistance. 
Supported by Grant EY05612 from the National Institutes of Health (Bethesda, Maryland, United States) and grants from Alcon Research, Ltd. (Houston, Texas, USA), the Margaret S. Sinon Scholar in Ocular Surface Research Fund, and the Guoxing Yao & Yang Liu Research Fund. 
Disclosure: D.A. Sullivan, Alcon Research, Ltd. (F); Y. Liu, None; W.R. Kam, None; J. Ding, None; K.M. Green, None; S.A. Shaffer, None; M.P. Hatton, None; S. Liu, None 
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Figure
 
Lipid accumulation in immortalized human meibomian gland epithelial cells. Cells were cultured in serum for 5 or 13 days, and then processed for the identification of neutral lipids with LipidTOX, lysosomes with LysoTracker Red DND-99, and nuclei with DAPI. The figures were overlaid with Adobe Photoshop (Adobe Systems, San Jose, CA, USA) to create the “Merge” figure.
Figure
 
Lipid accumulation in immortalized human meibomian gland epithelial cells. Cells were cultured in serum for 5 or 13 days, and then processed for the identification of neutral lipids with LipidTOX, lysosomes with LysoTracker Red DND-99, and nuclei with DAPI. The figures were overlaid with Adobe Photoshop (Adobe Systems, San Jose, CA, USA) to create the “Merge” figure.
Table 1
 
Influence of Serum on Gene Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 1
 
Influence of Serum on Gene Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Group Genes ↑ Genes ↓ Total Genes
Undiff vs. serum
 Primary 2125 2358 4483
 Immortalized 2025 2787 4812
SFM vs. serum
 Primary 1368 1814 3182
 Immortalized 1602 1187 2789
Table 2
 
Effect of Serum on Gene Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 2
 
Effect of Serum on Gene Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Accession # Gene Ratio Ontology
Upregulation
 NM_000478 Alkaline phosphatase 24.7, 11.9, 12.3, 11.9 Anatomical structure morphogenesis
 NM_019846 chemokine (C-C motif) ligand 28 15.7, 19.1, 18.9, 17.6 Chemotaxis
 NM_000598 Insulin-like growth factor binding protein 3 54.2, 33.8, 41.3, 20.8 Regulation of cell growth
 NM_002426 Matrix metallopeptidase 12 50.1, 45.6, 43.0, 54.8 Proteolysis
 NM_007069 Phospholipase A2, group XVI 55.4, 17.9, 24.3, 9.0 Lipid catabolic process
 NM_006017 Prominin 1 32.4, 31.9, 32.4, 31.9 Cell differentiation
 NM_206963 Retinoic acid receptor responder (tazarotene induced) 1 456.0, 207.0, 30.6, 99.5 Negative regulation of cell proliferation
 NM_019554 S100 calcium binding protein A4 24.1, 20.8, 12.3, 9.8 Epithelial to mesenchymal transition
 NM_003357 Secretoglobin, family 1A, member 1 31.2, 18.3, 29.3, 17.7 Negative regulation of transcription from RNA polymerase II promoter
 NM_030754 Serum amyloid A2 140.9, 32.2, 7.2, 43.6 Acute phase response
 NM_003186 Transgelin 62.0, 107.5, 8.1, 11.9 Anatomical structure development
Downregulation
 NM_001218 Carbonic anhydrase XII 11.9, 9.8, 8.8, 14.4 1-Carbon metabolic process
 NM_002775 HtrA serine peptidase 1 5.7, 6.5, 4.9, 7.5 Negative regulation of TGF β receptor signaling pathway
Table 3
 
Effect of Serum Exposure on Cellular Component Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 3
 
Effect of Serum Exposure on Cellular Component Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Ontology Undifferentiated vs. Serum SFM vs. Serum
Pri Genes Pri z-Score Imm Genes Imm z-Score Pri Genes Pri z-Score Imm Genes Imm z-Score
Upregulation
 Cytoplasmic membrane-bounded vesicle 97 3.14 89 2.99 68 3.2 81 4.07
 Cytoplasmic vesicle 105 3.5 101 3.94 72 3.29 89 4.64
 Endomembrane system 213 5.4 183 3.99 161 6.65 176 6.57
 Endoplasmic reticulum 144 4.14 120 2.6 105 4.69 132 6.7
 Endoplasmic reticulum membrane 90 3.41 81 3.03 71 4.69 81 5.17
 Endoplasmic reticulum part 101 3.55 86 2.51 82 5.27 95 6.02
 Golgi apparatus 162 7.25 143 6.26 107 5.9 128 7.26
 Golgi apparatus part 106 6.71 88 5.04 68 5.1 84 6.65
 Golgi cisterna 16 2.96 15 2.95 13 3.5 16 4.32
 Golgi cisterna membrane 13 2.51 11 2 10 2.73 13 3.72
 Golgi lumen 6 2.48 6 2.72 4 2.04 5 2.56
 Golgi membrane 84 5.56 73 4.65 61 5.56 72 6.53
 Golgi stack 24 4.29 20 3.41 18 4.31 21 4.87
 Lysosome 65 7.66 54 6.11 31 3.24 30 2.31
 Lytic vacuole 65 7.66 54 6.11 31 3.24 30 2.31
 Membrane-bounded vesicle 100 3.32 92 3.2 70 3.34 84 4.33
 Vacuole 69 8.26 69 7.73 38 3.88 34 2.25
 Vesicle 111 3.88 106 4.23 76 3.59 94 5.02
Downregulation
 Cytosolic large ribosomal subunit 28 12.9 22 8.67 29 15.54 15 9.42
 Cytosolic small ribosomal subunit 29 14.19 20 8.2 32 18.23 13 8.44
 Large ribosomal subunit 20 10.75 17 7.96 13 7.55 5 3
 Mitochondrial envelope 84 5.93 84 4.31 69 5.78 37 2.8
 Mitochondrial large ribosomal subunit 11 7.27 9 5.06 7 4.94 4 3.29
 Mitochondrial nucleoid 15 6.37 11 3.52 7 2.62 5 2.38
 Mitochondrial part 149 10.63 151 8.66 112 8.65 51 2.84
 Mitochondrion 265 12.63 292 12.05 202 10.51 102 4.34
 Organellar large ribosomal subunit 11 7.27 9 5.06 7 4.94 4 3.29
 Ribonucleoprotein complex 220 24.93 201 19.29 163 20.3 60 6.66
 Ribosome 113 22.93 93 16.03 98 22.68 35 8.33
Table 4
 
Influence of Serum on Biological Process Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 4
 
Influence of Serum on Biological Process Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Ontology Undifferentiated vs. Serum SFM vs. Serum
Pri Genes Pri z-Score Imm Genes Imm z-Score Pri Genes Pri z-Score Imm Genes Imm z-Score
Upregulation
 Anatomical structure development 349 2.64 334 3.35 256 4.28 291 4.4
 Apoptosis 195 6 184 6.13 132 5.21 159 6.43
 Apoptotic mitochondrial changes 11 3.68 9 2.89 10 4.69 8 2.97
 Cell adhesion 117 4.49 96 2.78 97 6.52 103 5.88
 Cell death 209 5.85 199 6.17 142 5.16 166 5.88
 Cell differentiation 237 2.25 223 2.49 162 2.38 195 3.38
 Cell migration 87 4.13 76 3.31 62 4.11 71 4.4
 Cell morphogenesis 86 2.81 75 2.07 62 3.11 73 3.64
 Cellular response to stimulus 500 4.25 446 3.07 337 3.99 387 4.31
 Epidermis development 42 4.72 46 6.23 28 3.86 27 2.82
 Epithelium development 65 4.3 61 4.28 46 4.09 49 3.67
 Induction of apoptosis 60 3.99 59 4.47 36 2.44 54 5.16
 Induction of programmed cell death 60 3.95 59 4.43 37 2.62 54 5.11
 Organ morphogenesis 76 2.24 75 2.84 53 2.29 72 4.24
 Programmed cell death 196 5.99 185 6.12 134 5.36 160 6.45
 Proteolysis 132 5.22 130 5.98 87 4.18 108 5.59
 Signal transduction 430 4.31 391 3.71 294 4.3 328 3.95
 Signaling 453 3.21 412 2.65 314 3.77 347 3.11
 Tissue development 136 5.27 128 5.33 98 5.39 111 5.61
 Vesicle-mediated transport 115 4.36 103 3.76 77 3.67 80 2.79
 Wound healing 84 3.67 71 2.55 59 3.56 75 5.01
Downregulation
 Cell cycle 209 7.95 327 15.98 124 2.54 84 2.34
 Cell cycle checkpoint 56 7.04 77 9.92 28 2.25 22 2.85
 Cell cycle phase 154 9.09 248 17.46 79 2.22 54 2.11
 Cell cycle process 168 8 275 16.69 95 2.37 64 2.12
 Cell division 79 6.01 139 13.58 47 2.47 37 3.32
 Gene expression 505 9.8 555 8.19 412 9.61 226 3.75
 M phase 92 6.38 172 15.94 52 2.12 37 2.26
 M phase of mitotic cell cycle 78 7.33 143 16.41 40 2.2 29 2.39
 Mitosis 76 7.31 139 16.26 39 2.23 29 2.59
 Mitotic cell cycle 151 10.08 233 17.53 76 2.72 50 2.17
 NcRNA metabolic process 93 12.6 96 11.27 71 10.55 32 4.49
 NcRNA processing 66 10.51 68 9.37 54 9.73 25 4.45
 Nuclear division 76 7.31 139 16.26 39 2.23 29 2.59
 Nuclear export 39 9.3 30 5.31 30 7.88 14 3.76
 Ribonucleoprotein complex assembly 33 8.13 30 6.03 19 4.44 10 2.38
 Ribonucleoprotein complex biogenesis 91 15.81 80 11.5 70 13.43 27 4.84
 Ribonucleoprotein complex subunit organization 34 8.06 31 5.97 20 4.52 11 2.63
 Ribonucleotide metabolic process 18 4.82 17 3.63 58 3.28 36 2.17
 Ribosome biogenesis 69 15.54 60 11.38 61 15.71 20 4.84
 RNA processing 172 13.95 180 12.43 126 10.82 52 3.11
 rRNA metabolic process 47 11.72 44 9.39 41 11.7 17 4.98
 rRNA processing 46 11.88 43 9.53 40 11.8 17 5.24
 Sterol biosynthetic process 19 6.39 19 5.52 9 2.52 10 4.56
 Translation 180 21.45 157 15.28 151 20.43 67 9.34
 Translational elongation 75 19.97 55 12.1 77 23.76 32 11.05
 Translational initiation 27 7.6 26 6.21 23 7.41 12 4.27
 Translational termination 68 19.2 52 12.31 74 24.37 31 11.52
Table 5
 
Impact of Serum on Molecular Function Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 5
 
Impact of Serum on Molecular Function Ontologies in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Ontology Undifferentiated vs. Serum SFM vs. Serum
Pri Genes Pri z-Score Imm Genes Imm z-Score Pri Genes Pri z-Score Imm Genes Imm z-Score
Upregulation
 Cysteine-type endopeptidase activity 13 2.74 12 2.57 9 2.46 13 3.9
 Cytoskeletal protein binding 70 2.76 62 2.13 58 4.49 56 2.99
 Enzyme binding 94 2.92 93 3.49 68 3.42 72 2.75
 Growth factor activity 23 3.8 17 2.19 23 6.15 21 2.78
 Peptidase activity 78 3.38 84 4.91 54 3.29 68 4.5
Downregulation
 DNA-dependent ATPase activity 16 3.38 25 5.94 12 2.73 8 2.17
 Electron carrier activity 31 3.28 31 2.29 24 2.8 16 2.21
 GTP binding 60 3.67 64 2.92 49 3.61 29 2
 GTPase activity 44 4.76 41 2.95 29 2.81 23 3.32
 Ligase activity 64 2.46 83 3.77 57 3.36 37 2.43
 mRNA binding 25 6.13 24 4.84 21 5.96 9 2.28
 Nucleic acid binding 437 7.33 492 6.55 351 7.1 211 3.51
 Nucleotide binding 345 8.25 389 7.76 250 5.57 150 2.67
 Purine nucleotide binding 257 4.97 310 5.94 191 3.46 126 2.52
 Purine ribonucleoside triphosphate binding 253 5.06 309 6.32 187 3.44 126 2.8
 Purine ribonucleotide binding 255 4.93 309 6 189 3.38 126 2.6
 Ribonucleotide binding 255 4.93 309 6 189 3.38 126 2.6
 RNA binding 251 19.9 223 13.73 198 17.62 89 7.18
 Structural constituent of ribosome 109 24.09 86 16.09 99 25.05 38 10.23
 Structure-specific DNA binding 36 3.92 44 4.63 32 4.46 18 2.47
 Transferase activity, transferring alkyl or aryl (other than methyl) groups 12 3.17 12 2.54 13 4.65 7 2.67
 Translation factor activity, nucleic acid binding 33 8.51 32 7.05 25 7.12 12 3.46
Table 6
 
Serum-Induced Alteration of Genes Related to Tissue Development in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 6
 
Serum-Induced Alteration of Genes Related to Tissue Development in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Accession # Gene Ratio Ontology
Upregulation
 NM_000598 Insulin-like growth factor binding protein 3 54.2, 33.8, 41.3, 20.8 Regulation of cell growth
 NM_005046 Kallikrein-related peptidase 7 52.5, 80.9, 4.5, 2.9 Epidermis development
 NM_001878 Cellular retinoic acid binding protein 2 44.8, 20.9, 2.0, 3.8 Epidermis development
 NM_000900 Matrix Gla protein 39.2, 3.5, 33.2, 4.8 Protein complex assembly
 NM_001323 Cystatin E/M 31.3, 59.2, 3.2, 5.6 Epidermis development
 NM_002167 Inhibitor of DNA binding 3 21.5, 28.5, 3.3, 5.5 Negative regulation of transcription
 NM_021229 Netrin 4 21.1, 4.2, 6.3, 3.7 Neuron remodeling
 NM_172037 Retinol dehydrogenase 10 18.0, 5.2, 8.4, 3.3 Organ morphogenesis
 NM_019554 S100 calcium binding protein A4 17.5, 20.8, 8.4, 3.5 Cell differentiation
 NM_002166 Inhibitor of DNA binding 2 14.9, 36.8, 5.3, 3.1 Negative regulation of transcription
 NM_004936 Cyclin-dependent kinase inhibitor 2B 14.4, 15.9, 5.0, 3.7 Regulation of cyclin-dependent protein kinase activity
 NM_001901 Connective tissue growth factor 14.0, 9.2, 4.6, 10.5 Angiogenesis
 NM_001453 Forkhead box C1 11.4, 8.8, 3.2, 3.1 Blood vessel development
 NM_000393 Collagen, type V, α2 7.6, 3.9, 2.5, 8.8 Axon guidance
 NM_001955 Endothelin 1 6.1, 4.1, 2.0, 4.1 Prostaglandin biosynthetic process
 NM_001018004 Tropomyosin 1α 4.6, 8.4, 2.1, 6.0 Cellular component movement
 NM_000693 Aldehyde dehydrogenase 1 family, member A3 2.9, 2.5, 3.1, 3.5 Retinoic acid biosynthetic process
 NM_000362 TIMP metallopeptidase inhibitor 3 2.2, 4.6, 2.8, 2.7 Central nervous system development
 NM_001039348 EGF-containing fibulin-like extracellular matrix protein 1 2.1, 2.4, 1.9, 12.6 Epidermal growth factor receptor signaling pathway
 NM_000963 Prostaglandin-endoperoxide synthase 2 2.0, 7.2, 5.6, 13.7 Prostaglandin biosynthetic process
Downregulation
 NM_003068 Snail homolog 2 5.6, 4.2, 3.1, 2.2 Negative regulation of transcription
 NM_005213 Cystatin A 5.2, 3.2, 4.3, 16.9 Negative regulation of peptidase activity
 NM_000575 Interleukin 1α 4.5, 3.6, 2.2, 2.6 Inflammatory response
 NM_033260 Forkhead box Q1 4.2, 5.2, 2.5, 3.7 DNA fragmentation involved in apoptotic nuclear change
 NM_006142 Stratifin 4.1, 2.3, 2.4, 2.0 Regulation of cyclin-dependent protein kinase activity
 NM_000067 Carbonic anhydrase II 4.0, 2.6, 13.3, 7.3 1-Carbon metabolic process
 NM_004429 Ephrin-B1 4.0, 2.9, 2.2, 2.2 Neural crest cell migration
Table 7
 
Effect of Serum on KEGG Pathways in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 7
 
Effect of Serum on KEGG Pathways in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Ontology Undifferentiated vs. Serum SFM vs. Serum
Pri Genes Pri z-Score Imm Genes Imm z-Score Pri Genes Pri z-Score Imm Genes Imm z-Score
Upregulation
 Lysosome 35 6.5 31 5.96 21 4.29 17 2.29
 p53 signaling pathway 18 4.15 19 5.05 12 3.26 16 4.51
 Phagosome 37 5.54 31 4.57 25 4.45 26 4.02
Downregulation
 Aminoacyl-tRNA biosynthesis 12 3.1 17 4.96 11 3.58 7 2.82
 Cell cycle 36 5.3 51 8.51 24 3.43 21 4.83
 Ribosome 69 18.22 50 11.32 74 23.08 31 11.08
 RNA transport 51 7.58 57 8.19 40 6.71 17 2.45
 Steroid biosynthesis 10 5.04 10 4.66 6 3.05 6 4.43
Table 8
 
Confirmation of Selected BeadChip Gene Expression Results
Table 8
 
Confirmation of Selected BeadChip Gene Expression Results
Gene Cell SFM BeadChip SFM qPCR Serum BeadChip Serum qPCR
Upregulation
 LCN2 Primary 6.2 7.5 72.0 161.9
 LCN2 Imm 22.6 34.0 21.7 40.3
 CLDN1 Primary 15.1 13.9 10.2 11.5
 CLDN1 Imm 8.1 9.0 8.6 9.8
Downregulation
 ODC1 Primary 7.8 12.1 21.9 19.7
 ODC1 Imm 4.5 5.9 6.9 14.6
 ACAT2 Primary 10.3 15.4 12.4 17.4
 ACAT2 Imm 4.3 3.4 7.1 9.5
Table 9
 
Influence of Serum on Wax and Cholesterol Ester Fatty Acid Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Table 9
 
Influence of Serum on Wax and Cholesterol Ester Fatty Acid Expression in Primary and Immortalized Human Meibomian Gland Epithelial Cells
Fatty Acid Serum-Free Media Serum-Containing Media
Primary Cells Immortalized Cells Primary Cells Immortalized Cells
Wax ester
 33:1 587.0, 476.6 707.2, 897.2 166.7, 115.8 291.8, 236.1
 41:3 58.0, 41.9 24.3, 25.2 162.9, 92.4 78.7, 121.5
 42:2 56.4, 84.2 37.7, 67.2 234.5, 205.0 291.4, 292.7
Cholesterol ester
 20:3 89.0, 216.2 166.2, 176.8 854.7, 1023.9 818.1, 1216.9
 20:4* 70.4, 364.5* 220.6, 470.3* 744.7, 1152.0* 887.9, 1192.5*
 22:4 1.0, 8.5 6.8, 5.7 148.0, 173.7 154.8, 279.6
Table 10
 
Phospholipid Expression in Human Primary and Immortalized Meibomian Gland Epithelial Cells In Vitro
Table 10
 
Phospholipid Expression in Human Primary and Immortalized Meibomian Gland Epithelial Cells In Vitro
Phospholipid Serum-Free Media Serum-Containing Media
Primary Cells Immortalized Cells Primary Cells Immortalized Cells
Phosphati-dylcholine 57.00, 52.35 49.43, 43.93 55.97, 50.63 47.04, 44.97
Sphingomyelin 19.93, 16.91 20.55, 20.47 13.49, 15.45 18.47, 17.95
Lysophosphati-dylethanolamine 11.24, 13.16 11.98, 15.48 14.13, 15.30 14.77, 16.89
Lysophospha-tidylserine 3.70, 5.01 5.61, 7.03 6.45, 6.76 7.24, 7.97
Phosphatidyl-serine 2.39, 4.69 4.17, 4.53 4.05, 5.27 5.47, 4.91
Phosphatidyl-ethanolamine 2.47, 3.63 3.29, 3.05 2.59, 3.60 3.02, 2.68
Lysophosphati-dylglycerol 1.55, 2.26 2.46, 2.55 1.94, 1.29 1.51, 2.67
Lysophosphatidic acid 1.27, 1.17 1.41, 1.46 0.91, 1.12 1.57, 1.22
Phosphatidic acid 0.41, 0.70 0.97, 1.31 0.41, 0.51 0.84, 0.63
Phosphati-dylglycerol 0.05, 0.13 0.13, 0.19 0.08, 0.07 0.07, 0.10
Table 11
 
Effect of Serum Exposure on Phospholipid Fatty Acid Content in Human Primary and Immortalized Meibomian Gland Epithelial Cells
Table 11
 
Effect of Serum Exposure on Phospholipid Fatty Acid Content in Human Primary and Immortalized Meibomian Gland Epithelial Cells
Phospholipid Serum-Free Media Serum-Containing Media
Primary Cells Immortalized Cells Primary Cells Immortalized Cells
Phosphatidylcholine
 38:2 2480, 2976 2798, 3794 1,795, 828.2 1,687, 1,168
 38:4* 567.2, 1206* 699.4, 647.2* 14,642, 9,083* 5,157, 5,304*
 38:5 277.4, 629.3 338.3, 292.5 10,131, 6,107 4,206, 4,535
 38:6 97.2, 156.1 66.0, 59.9 2,565, 1,674 1,169, 1,115
Lysophosphatidylethanolamine
 20:4* 2155, 5363* 2192, 3504* 19,861, 12,622* 11,603, 15,859*
 22:2 204.2, 386.6 364.0, 1091 75.6, 45.7 104.5, 77.5
 22:4 312.6, 811.0 585.6, 1457 3,154, 2,254 2,220, 3,016
 22:6 456.0, 1080 479.1, 916.8 21,845, 16,015 12,697, 14,728
Lysophosphatidylserine
 22:5 193.3, 521.9 240.6, 481.3 2,080, 1,383 1,449, 1,585
 22:6 75.8, 222.9 103.6, 198.4 3,872, 2,435 2,111, 2,756
Phosphatidylserine
 40:6 56.7, 235.0 107.5, 160.8 2,836, 2,301 1,866, 1,897
 40:7 17.8, 51.3 66.7, 25.0 221.3, 213.1 164.3, 131.4
 42:9 0.4, 1.8 0.5, 0.8 118.8, 104.9 81.9, 79.6
Phosphatidylethanolamine
 38:2 312.8, 569.5 440.2, 618.0 167.0, 142.5 203.1, 118.4
 38:4* 330.0, 975.4* 365.8, 404.6* 2,970, 2,792* 1,810, 1,753*
 38:6 30.7, 73.2 26.1, 26.9 551.7, 571.7 329.5, 321.0
 40:7 17.0, 49.9 35.3, 35.3 925.7, 898.9 685.5, 616.5
Lysophosphatidylglycerol
 20:2 212.4, 823.6 585.1, 973.0 3,608, 1,574 1,550, 3,348
 20:4* 39.2, 131.8* 119.3, 155.1* 642.5, 277.9* 232.3, 569.1*
 22:5 21.8, 85.0 24.7, 39.6 530.4, 205.6 232.2, 490.5
 22:6 19.3, 75.2 18.8, 37.2 2,261, 1,029 970.4, 2,117
Phosphatidylglycerol
 40:7 0.6, 4.8 1.7, 3.9 117.1, 53.6 51.7, 97.0
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