Cytoprotective Effects of Human Platelet Lysate during the Xeno-Free Culture of Human Donor Corneas
Abstract
:1. Introduction
2. Results
2.1. 2%HPL Led to a Lower Endothelial Cell Loss Than 2%FBS
2.2. 2%HPL and 2%FBS Did Not Influence Endothelial Cell Morphology
2.3. NGS Indicated a More Extensive and Robust Differential Gene Regulation in Endothelial Compared to Stromal Corneal Cells
2.4. Differential 2%HPL and 2%FBS Culture Did Not Influence the Expression Levels of Known Corneal Stromal Cell Markers, but Caused Mild Alterations among Corneal Endothelial Cell Markers
2.5. Category Netplots Visualize That Gene Expression Alterations following 2%HPL vs. 2%FBS Cornea Culture Influenced Different Areas of Cell Function in Stromal Compared to Corneal Endothelial Cells
2.6. GSEA (Gene Set Enrichment Analysis) of Hallmark Gene Sets and Associated DGEA (Differential Gene Expression Analysis) Showed Differential Expression Patterns Predominantly in the Corneal Endothelial Cells
3. Discussion
4. Material and Methods
4.1. Organ Culture and Cornea Processing
4.2. Endothelial Cell Density and Morphology Evaluation
4.3. Cornea Processing for NGS
4.4. Next Generation Sequencing
4.5. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Donor | Gender | Age (Years) | Death to Cornea Collection Time (Hours) | Cause of Death |
---|---|---|---|---|
1 | f | 79 | 61.17 | hemorrhagic shock |
2 | m | 59 | 59.18 | hemorrhagic shock; thoracoabdominal aneurysm of Aorta |
3 | m | 61 | 38.17 | multiple organ failure; liver cirrhosis |
4 | f | 81 | 14.25 | multiple organ failure; hepatic failure |
5 | m | 64 | 71.87 | multiple organ failure; liver cirrhosis |
6 | f | 85 | 25.30 | renal failure |
7 | f | 84 | 37.48 | respiratory failure; fall |
8 | f | 95 | 63.33 | basilar artery occlusion, ischemic stroke |
9 | f | 31 | 23.65 | pancreatic carcinoma |
10 | m | 62 | 39.85 | hemorrhagic shock; perforated coronary bypass |
11 | f | 54 | 32.17 | septic shock; acute myeloid leukemia |
12 | f | 57 | 30.17 | pancreatic carcinoma |
13 | m | 73 | 41.68 | cerebral hemorrhage |
14 | m | 67 | 32.13 | septic shock; carcinoma of colon |
15 | m | 72 | 17.07 | hepatic failure; bladder carcinoma |
16 | f | 84 | 28.58 | rectal carcinoma |
Mean | 69.25 | 38.50 | ||
SD | 15.72 | 17.08 |
Donor | Culture | ECD TP1 2%HPL Cells/mm2 | ECD TP2 2%HPL Cells/mm2 | ECD Change Cells/mm2 | ECD Change% | Culture | ECD TP1 2%FBS Cells/mm2 | ECD TP2 2%FBS Cells/mm2 | ECD Change Cells/mm2 | ECD Change % |
---|---|---|---|---|---|---|---|---|---|---|
1 | HPL | 1935 | 1995 | 60 | 3.10 | FBS | 1905 | 2040 | 135 | 7.09 |
2 | HPL | 2050 | 2055 | 5 | 0.24 | FBS | 2035 | 1950 | −85 | −4.18 |
3 | HPL | 2600 | 2560 | −40 | −1.54 | FBS | 2375 | 2295 | −80 | −3.37 |
4 | HPL | 2630 | 2590 | −40 | −1.52 | FBS | 2535 | 2515 | −20 | −0.79 |
5 | HPL | 1970 | 1940 | −30 | −1.52 | FBS | 1855 | 1840 | −15 | −0.81 |
6 | HPL | 2485 | 2440 | −45 | −1.81 | FBS | 2520 | 2465 | −55 | −2.18 |
7 | HPL | 1610 | 1570 | −40 | −2.48 | FBS | 1350 | 1310 | −40 | −2.96 |
8 | HPL | 1885 | 1870 | −15 | −0.80 | FBS | 1825 | 1750 | −75 | −4.11 |
9 | HPL | 2094 | 2088 | −6 | −0.29 | FBS | 2340 | 2125 | −215 | −9.19 |
10 | HPL | 2610 | 2525 | −85 | −3.26 | FBS | 2720 | 2560 | −160 | −5.88 |
11 | HPL | 2315 | 2400 | 85 | 3.67 | FBS | 2445 | 2255 | −190 | −7.77 |
12 | HPL | 2220 | 2125 | −95 | −4.28 | FBS | 2300 | 2230 | −70 | −3.04 |
13 | HPL | 2095 | 2105 | 10 | 0.48 | FBS | 2350 | 2200 | −150 | −6.38 |
14 | HPL | 2375 | 2325 | −50 | −2.11 | FBS | 2490 | 2285 | −205 | −8.23 |
15 | HPL | 2290 | 2335 | 45 | 1.97 | FBS | 2255 | 2255 | 0 | 0.00 |
16 | HPL | 1945 | 1935 | −10 | −0.51 | FBS | 1910 | 1725 | −185 | −9.69 |
MEAN | 2194.31 | 2178.63 | −15.69 | −0.67 | 2200.63 | 2112.50 | −88.13 | −3.84 | ||
SD | 297.35 | 288.14 | 48.90 | 2.17 | 355.47 | 330.99 | 93.11 | 4.21 |
Time Point | Polymegethism | Pleomorphism | Granulation | Vacuolization | Segmentation of Cell Membranes | Endothelial Cell-Free Areas | Descemet Folds | Trypan Blue-Positive Cells | ||
---|---|---|---|---|---|---|---|---|---|---|
2% HPL | 1 | Mean | 1.44 | 1.19 | 0.00 | 0.13 | 0.94 | 0.13 | 1.88 | 0.00 |
SD | 0.73 | 0.40 | 0.00 | 0.34 | 0.25 | 0.50 | 0.50 | 0.00 | ||
2% HPL | 2 | Mean | 1.38 | 1.25 | 0.00 | 0.06 | 1.00 | 0.00 | 2.13 | 0.00 |
SD | 0.62 | 0.45 | 0.00 | 0.25 | 0.00 | 0.00 | 0.50 | 0.00 | ||
2% FBS | 1 | Mean | 1.38 | 1.44 | 0.00 | 0.06 | 1.00 | 0.00 | 1.94 | 0.00 |
SD | 0.72 | 0.63 | 0.00 | 0.25 | 0.00 | 0.00 | 0.57 | 0.00 | ||
2% FBS | 2 | Mean | 1.50 | 1.31 | 0.00 | 0.13 | 0.94 | 0.00 | 2.06 | 0.00 |
SD | 0.63 | 0.60 | 0.00 | 0.34 | 0.25 | 0.00 | 0.44 | 0.00 |
Gene | Full Name | Molecular Function | Mean | log2FC | ||
---|---|---|---|---|---|---|
E N D O U P | 1 | GFRA1 | GDNF family receptor alpha 1 | glial cell-derived neurotrophic factor receptor activity | 10.67 | 4.46 |
2 | GFRA2 | GDNF family receptor alpha 2 | glial cell-derived neurotrophic factor receptor activity | 9.80 | 4.07 | |
3 | CST1 | cystatin SN | cysteine-type endopeptidase inhibitor activity | 50.55 | 3.97 | |
4 | S1PR5 | sphingosine-1-phosphate receptor 5 | G protein-coupled receptor activity | 23.90 | 3.89 | |
5 | C5orf46 | chromosome 5 open reading frame 46 | unclear | 46.64 | 3.86 | |
6 | DCX | doublecortin | microtubule binding, protein kinase binding | 11.45 | 3.81 | |
7 | LEFTY2 | left-right determination factor 2 | cytokine activity | 50.76 | 3.74 | |
8 | FNDC1 | fibronectin type III domain containing 1 | activator of G protein signaling | 159.43 | 3.67 | |
9 | NEURL1B | neuralized E3 ubiquitin protein ligase 1B | ubiquitin protein ligase activity | 28.72 | 3.45 | |
10 | HMOX1 | heme oxygenase 1 | protein homodimerization activity, oxidoreductase activity | 849.53 | 3.44 | |
E N D O D O W N | 1 | CACNA1F | calcium voltage-gated channel subunit alpha1 F | ion channel activity, high voltage-gated calcium channel activity | 5.11 | −2.90 |
2 | SIK1B | salt inducible kinase 1B | transferase activity, transferring phosphorus-containing groups, protein tyrosine kinase activity | 8.18 | −2.89 | |
3 | TTC23L | tetratricopeptide repeat domain 23 like | unclear | 8.26 | −2.76 | |
4 | CCDC201 | coiled-coil domain containing 201 | unclear | 5.13 | −2.70 | |
5 | LCN12 | lipocalin 12 | transporter activity, retinoic acid binding | 12.63 | −2.54 | |
6 | GALNT16 | polypeptide N-acetylgalactosaminyltransferase 16 | carbohydrate binding, polypeptide N-acetylgalactosaminyltransferase activity | 5.55 | −2.52 | |
7 | RANBP3L | RAN binding protein 3 like | SMAD binding activity | 118.52 | −2.49 | |
8 | B3GALT1 | Beta-1,3-galactosyltransferase 1 | galactosyltransferase activity, UDP-galactose:beta-N-acetylglucosamine beta-1,3-galactosyltransferase activity | 7.00 | −2.48 | |
9 | DCC | DCC netrin 1 receptor | transmembrane signaling receptor activity, axon guidance | 89.05 | −2.45 | |
10 | ETNPPL | polo like kinase 5 | regulatory kinase of cell cycle, apoptosis | 567.33 | −2.45 | |
S T R O M A U P | 1 | MKX | mohawk homeobox | sequence-specific DNA binding, DNA-binding transcription activator activity, RNA polymerase II-specific | 7.36 | 1.83 |
2 | ANGPTL4 | angiopoietin like 4 | enzyme inhibitor activity (inactivation of the lipoprotein lipase LPL) | 156.66 | 1.68 | |
3 | HMOX1 | heme oxygenase 1 | protein homodimerization activity, oxidoreductase activity | 428.25 | 1.35 | |
4 | PLIN2 | perilipin 2 | lipid globule surface membrane material | 2202.47 | 1.29 | |
5 | PDK4 | pyruvate dehydrogenase kinase 4 | protein kinase activity | 106.98 | 1.26 | |
6 | TFPI2 | tissue factor pathway inhibitor 2 | serine-type endopeptidase inhibitor activity, peptidase inhibitor activity | 78.66 | 1.25 | |
7 | PTX3 | pentraxin 3 | virion binding, (1->3)-beta-D-glucan binding | 705.56 | 1.24 | |
8 | ESM1 | endothelial cell specific molecule 1 | integrin binding, hepatocyte growth factor receptor binding | 50.20 | 1.12 | |
9 | LPXN | leupaxin | focal adhesion protein, cell type-specific signaling | 58.77 | 1.07 | |
10 | CBL | Cbl proto-oncogene | cell signaling and protein ubiquitination | 239.57 | 1.06 | |
S T R O M A D O W N | 1 | ANXA8L1 | annexin A8 like 1 | calcium ion binding, calcium-dependent phospholipid binding | 48.27 | −8.37 |
2 | PPP2R3B | protein phosphatase 2 regulatory subunit B’’beta | calcium ion binding, protein serine/threonine phosphatase activity | 19.56 | −4.37 | |
3 | SLURP1 | secreted LY6/PLAUR domain containing 1 | cytokine activity | 31.10 | −3.93 | |
4 | GJA4 | gap junction protein alpha 4 | gap junction channel activity | 11.78 | −3.42 | |
5 | LYPD2 | LY6/PLAUR domain containing 2 | post-translational modification: synthesis of GPI-anchored proteins | 15.07 | −3.33 | |
6 | CEACAM7 | CEA cell adhesion molecule 7 | Post-translational modification: synthesis of GPI-anchored proteins | 8.64 | −3.17 | |
7 | FSTL4 | follistatin like 4 | calcium ion binding | 7.43 | −2.90 | |
8 | PIGR | polymeric immunoglobulin receptor | immunoglobulin receptor activity (innate immune system) | 27.42 | −2.81 | |
9 | ANXA9 | annexin A9 | calcium ion binding, phospholipid binding | 15.02 | −2.80 | |
10 | PCDH19 | protocadherin 19 | calcium-dependent cell-adhesion protein | 42.01 | −2.51 |
Gene_Name | baseMean | log2FoldChange | padj | sig |
---|---|---|---|---|
Stroma | ||||
LUM | 7339.60 | −0.04 | 0.97 | Non-sig. |
ALDH3A1 | 5061.16 | −0.20 | 0.81 | Non-sig. |
ALDH1A1 | 360.23 | −0.04 | 0.96 | Non-sig. |
COL3A1 | 322.41 | −0.63 | 0.62 | Non-sig. |
KERA | 247.90 | −0.43 | 0.56 | Non-sig. |
THY1 | 119.26 | −0.06 | 0.96 | Non-sig. |
ACTA2 | 92.73 | 0.40 | 0.64 | Non-sig. |
TNC | 52.40 | 0.16 | 0.91 | Non-sig. |
Endothelium | ||||
PTGDS | 7862.98 | −1.27 | 0.0000 | Sig. genes |
AQP1 | 4592.68 | −0.26 | 0.3667 | Non-sig. |
ATP1A1 | 2506.61 | −0.69 | 0.0000 | Sig. genes |
SLC4A11 | 2153.22 | −1.24 | 0.0000 | Sig. genes |
COL8A2 | 1929.74 | −0.05 | 0.8879 | Non-sig. |
CDH2 | 1667.72 | 0.69 | 0.0000 | Sig. genes |
SLC4A4 | 1644.20 | −0.47 | 0.0406 | Sig. genes |
TJP1 | 579.02 | −0.27 | 0.3754 | Non-sig. |
ENO2 | 578.72 | 0.10 | 0.7347 | Non-sig. |
VDAC2 | 532.68 | −0.14 | 0.1763 | Non-sig. |
VDAC3 | 522.33 | −0.25 | 0.0629 | Non-sig. |
CLCN3 | 475.47 | 0.16 | 0.5404 | Non-sig. |
ACTA2 | 178.71 | 0.90 | 0.1877 | Non-sig. |
F11R | 100.05 | −0.19 | 0.6834 | Non-sig. |
CLCN2 | 9.39 | −0.29 | 0.7173 | Non-sig. |
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Talpan, D.; Salla, S.; Meusel, L.; Walter, P.; Kuo, C.-C.; Franzen, J.; Fuest, M. Cytoprotective Effects of Human Platelet Lysate during the Xeno-Free Culture of Human Donor Corneas. Int. J. Mol. Sci. 2023, 24, 2882. https://doi.org/10.3390/ijms24032882
Talpan D, Salla S, Meusel L, Walter P, Kuo C-C, Franzen J, Fuest M. Cytoprotective Effects of Human Platelet Lysate during the Xeno-Free Culture of Human Donor Corneas. International Journal of Molecular Sciences. 2023; 24(3):2882. https://doi.org/10.3390/ijms24032882
Chicago/Turabian StyleTalpan, Delia, Sabine Salla, Linus Meusel, Peter Walter, Chao-Chung Kuo, Julia Franzen, and Matthias Fuest. 2023. "Cytoprotective Effects of Human Platelet Lysate during the Xeno-Free Culture of Human Donor Corneas" International Journal of Molecular Sciences 24, no. 3: 2882. https://doi.org/10.3390/ijms24032882