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Nucleus Morphometry in Cultured Epithelial Cells Correlates with Phenotype

  • Ayyad Z. Khan (a1) (a2), Tor P. Utheim (a2) (a3) (a4) (a5), Catherine J. Jackson (a2), Sjur Reppe (a2), Torstein Lyberg (a2) and Jon R. Eidet (a2) (a6)...

Phenotype of cultured ocular epithelial transplants has been shown to affect clinical success rates following transplantation to the cornea. The purpose of this study was to evaluate the relationship between cell nucleus morphometry and phenotype in three types of cultured epithelial cells. This study provides knowledge for the development of a non-invasive method of determining the phenotype of cultured epithelium before transplantation. Cultured human conjunctival epithelial cells (HCjE), human epidermal keratinocytes (HEK), and human retinal pigment epithelial cells (HRPE) were analyzed by quantitative immunofluorescence. Assessments of nucleus morphometry and nucleus-to-cytoplasm ratio (N/C ratio) were performed using ImageJ. Spearman’s correlation coefficient was employed for statistical analysis. Levels of the proliferation marker PCNA in HCjE, HEK, and HRPE correlated positively with nuclear area. Nuclear area correlated significantly with levels of the undifferentiated cell marker ABCG2 in HCjE. Bmi1 levels, but not p63α levels, correlated significantly with nuclear area in HEK. The N/C ratio did not correlate significantly with any of the immunomarkers in HCjE (ABCG2, CK7, and PCNA) and HRPE (PCNA). In HEK, however, the N/C ratio was negatively correlated with levels of the undifferentiated cell marker CK14 and positively correlated with Bmi1 expression. The size of the nuclear area correlated positively with proliferation markers in all three epithelia. Morphometric indicators of phenotype in cultured epithelia can be identified using ImageJ. Conversely, the N/C ratio did not show a uniform relationship with phenotype in HCjE, HEK, or HRPE. N/C ratio therefore, may not be a useful morphometric marker for in vitro assessment of phenotype in these three epithelia.

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Al-Janabi, S., Huisman, A. & Van Diest, P.J. (2012). Digital pathology: Current status and future perspectives. Histopathology 61(1), 19.
Ang, L.P., Tan, D.T., Cajucom-Uy, H., Phan, T.T., Beuerman, R.W. & Lavker, R.M. (2004). Reconstruction of the ocular surface by transplantation of a serum free cultivated conjunctival tissue equivalent. Ann Acad Med Singapore 33(5 Suppl), S55S56.
Ang, L.P., Tanioka, H., Kawasaki, S., Ang, L.P., Yamasaki, K., Do, T.P., Thein, Z.M., Koizumi, N., Nakamura, T., Yokoi, N., Komuro, A., Inatomi, T., Nakatsukasa, M. & Kinoshita, S. (2010). Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency. Invest Ophthalmol Vis Sci 51(2), 758764.
Angello, J.C., Pendergrass, W.R., Norwood, T.H. & Prothero, J. (1987). Proliferative potential of human fibroblasts: An inverse dependence on cell size. J Cell Physiol 132(1), 125130.
Arpitha, P., Prajna, N.V., Srinivasan, M. & Muthukkaruppan, V. (2005). High expression of p63 combined with a large N/C ratio defines a subset of human limbal epithelial cells: Implications on epithelial stem cells. Invest Ophthalmol Vis Sci 46(10), 36313636.
Arpitha, P., Prajna, N.V., Srinivasan, M. & Muthukkaruppan, V. (2008 a). A method to isolate human limbal basal cells enriched for a subset of epithelial cells with a large nucleus/cytoplasm ratio expressing high levels of p63. Microsc Res Tech 71(6), 469476.
Arpitha, P., Prajna, N.V., Srinivasan, M. & Muthukkaruppan, V. (2008 b). A subset of human limbal epithelial cells with greater nucleus-to-cytoplasm ratio expressing high levels of p63 possesses slow-cycling property. Cornea 27(10), 11641170.
Barrandon, Y. & Green, H. (1985). Cell size as a determinant of the clone-forming ability of human keratinocytes. Proc Natl Acad Sci USA 82(16), 53905394.
Budak, M.T., Alpdogan, O.S., Zhou, M., Lavker, R.M., Akinci, M.A. & Wolosin, J.M. (2005). Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells. J Cell Sci 118(Pt 8), 17151724.
Cavalier-Smith, T. (2005). Economy, speed and size matter: Evolutionary forces driving nuclear genome miniaturization and expansion. Ann Bot 95(1), 147175.
Chen, Z., De Paiva, C.S., Luo, L., Kretzer, F.L., Pflugfelder, S.C. & Li, D.Q. (2004). Characterization of putative stem cell phenotype in human limbal epithelia. Stem Cells 22(3), 355366.
Cornish, T.C., Swapp, R.E. & Kaplan, K.J. (2012). Whole-slide imaging: Routine pathologic diagnosis. Adv Anat Pathol 19(3), 152159.
Da Cruz, L., Chen, F.K., Ahmado, A., Greenwood, J. & Coffey, P. (2007). RPE transplantation and its role in retinal disease. Prog Retin Eye Res 26(6), 598635.
Dazard, J.E., Piette, J., Basset-Seguin, N., Blanchard, J.M. & Gandarillas, A. (2000). Switch from p53 to MDM2 as differentiating human keratinocytes lose their proliferative potential and increase in cellular size. Oncogene 19(33), 36933705.
Eidet, J.R., Pasovic, L., Maria, R., Jackson, C.J. & Utheim, T.P. (2014). Objective assessment of changes in nuclear morphology and cell distribution following induction of apoptosis. Diagn Pathol 9, 92.
Falbo, K.B. & Shen, X. (2006). Chromatin remodeling in DNA replication. J Cell Biochem 97(4), 684689.
Gao, F.B. & Raff, M. (1997). Cell size control and a cell-intrinsic maturation program in proliferating oligodendrocyte precursor cells. J Cell Biol 138(6), 13671377.
Gregory, T.R. (Ed.) (2005). Genome size evolution in animals, In The Evolution of the Genome, pp. 4–87. San Diego: Elsevier.
Harris, H. (1967). The reactivation of the red cell nucleus. J Cell Sci 2(1), 2332.
Jara-Lazaro, A.R., Thamboo, T.P., Teh, M. & Tan, P.H. (2010). Digital pathology: Exploring its applications in diagnostic surgical pathology practice. Pathology 42(6), 512518.
Le Beyec, J., Xu, R., Lee, S.Y., Nelson, C.M., Rizki, A., Alcaraz, J. & Bissell, M.J. (2007). Cell shape regulates global histone acetylation in human mammary epithelial cells. Exp Cell Res 313(14), 30663075.
Lyngholm, M., Vorum, H., Nielsen, K., Ostergaard, M., Honore, B. & Ehlers, N. (2008). Differences in the protein expression in limbal versus central human corneal epithelium – A search for stem cell markers. Exp Eye Res 87(2), 96105.
Mao, Y. (2012). Nearest Neighbor Distances calculation with ImageJ (updated 4 January 2012) Available at (retrieved June 23, 2015).
Marchini, G., Pedrotti, E., Pedrotti, M., Barbaro, V., Di Iorio, E., Ferrari, S., Bertolin, M., Ferrari, B., Passilongo, M., Fasolo, A. & Ponzin, D. (2012). Long-term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency due to chemical burns. Clin Experiment Ophthalmol 40(3), 255267.
Meyer-Blazejewska, E.A., Kruse, F.E., Bitterer, K., Meyer, C., Hofmann-Rummelt, C., Wunsch, P.H. & Schlotzer-Schrehardt, U. (2010). Preservation of the limbal stem cell phenotype by appropriate culture techniques. Invest Ophthalmol Vis Sci 51(2), 765774.
Nazeran, H., Rice, F., Moran, W. & Skinner, J. (1995). Biomedical image processing in pathology: A review. Australas Phys Eng Sci Med 18(1), 2638.
Neumann, F.R. & Nurse, P. (2007). Nuclear size control in fission yeast. J Cell Biol 179(4), 593600.
Pantanowitz, L. (2010). Digital images and the future of digital pathology. J Pathol Inform 1, 115.
Parsa, R., Yang, A., McKeon, F. & Green, H. (1999). Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 113(6), 10991105.
Pasovic, L., Eidet, J., Lyberg, T., Messelt, E., Aabel, P. & Utheim, T. (2014). Antioxidants improve the viability of stored adult retinal pigment Epithelial-19 cultures. Ophthalmol Ther (3), 4961.
Pellegrini, G., Dellambra, E., Golisano, O., Martinelli, E., Fantozzi, I., Bondanza, S., Ponzin, D., McKeon, F. & De Luca, M. (2001). p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98(6), 31563161.
Pellegrini, G., Traverso, C.E., Franzi, A.T., Zingirian, M., Cancedda, R. & De Luca, M. (1997). Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349(9057), 990993.
Priya, C.G., Prasad, T., Prajna, N.V. & Muthukkaruppan, V. (2013). Identification of human corneal epithelial stem cells on the basis of high ABCG2 expression combined with a large N/C ratio. Microsc Res Tech 76(3), 242248.
Rama, P., Matuska, S., Paganoni, G., Spinelli, A., De Luca, M. & Pellegrini, G. (2010). Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 363(2), 147155.
Rocha, R., Vassallo, J., Soares, F., Miller, K. & Gobbi, H. (2009). Digital slides: Present status of a tool for consultation, teaching, and quality control in pathology. Pathol Res Pract 205(11), 735741.
Sonoda, S., Spee, C., Barron, E., Ryan, S.J., Kannan, R. & Hinton, D.R. (2009). A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells. Nat Protoc 4(5), 662673.
Tseng, H. & Green, H. (1994). Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation. J Cell Biol 126(2), 495506.
Utheim, T.P. (2013). Limbal epithelial cell therapy: Past, present, and future. Methods Mol Biol 1014, 343.
Vergani, L., Grattarola, M. & Nicolini, C. (2004). Modifications of chromatin structure and gene expression following induced alterations of cellular shape. Int J Biochem Cell Biol 36(8), 14471461.
Watt, F.M. & Green, H. (1981). Involucrin synthesis is correlated with cell size in human epidermal cultures. J Cell Biol 90(3), 738742.
Yang, X., Moldovan, N.I., Zhao, Q., Mi, S., Zhou, Z., Chen, D., Gao, Z., Tong, D. & Dou, Z. (2008). Reconstruction of damaged cornea by autologous transplantation of epidermal adult stem cells. Molecular Vision 14, 1064.
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Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
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