Nucleus Morphometry in Cultured Epithelial Cells Correlates with Phenotype

Abstract

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.

Determination of cell nucleus-to-cytoplasmic ratio using imaging flow cytometry and a combined ultrasound and photoacoustic technique: a comparison study

“…[4][5][6] These shortcomings have led to the development of alternative systems for N:C ratio quantification. Emerging technologies such as digital image analysis using HALO imaging-analysis software in urine cytology, 7 immunofluorescence microscopy paired with ImageJ, 8 and 3-D multiphoton microscopy 9 have been used to assess the N:C ratio but all require a lengthy imaging/analysis time for individual cells. Flow cytometry (FC) overcomes the drawback of slow analysis speeds at the expense of direct measurements of cell morphology.…”

Changes in the Cytomorphometric Indices in Epitheliocytes of the Oral Mucosa of Patients with Generalized Periodontitis

“…Thus, changes in the size of epithelial cells are important for the detection of cellular mechanisms in the pathogenesis of GP. It is reasonable to assert that the measurement of the parameters of epithelial cells in combination with the indices of their nuclei reflects the functional activity of the hereditary apparatus in GP, which was proven by scientists studying other human cells [22,23].…”

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins

“…The exposure length and gain were maintained at a constant level for all samples and the fluorescence intensities of the FITC or Cy3 fluorochromes, which were conjugated to the secondary antibodies, were within the dynamic range of the camera. Immunofluorescence per cell was quantified by custom-made macros for ImageJ, as described previously [ 7 , 19 ].…”