Epithelium, Anterior Corneal

HKCs were isolated from human corneas from patients with Keratoconus defects. These corneas were obtained from Ula Jurkunas (Massachusetts Eye and Ear Infirmary, Boston, MA, USA). HCFs were isolated from normal human corneas obtained from NDRI (National Disease Research Interchange; Philadelphia, PA). Briefly, corneal epithelium and endothelium were removed from the stroma by scraping with a razor blade. The stromal tissue was cut into small pieces (~ 2 × 2mm) and put into 6-well plates (4 or 5 pieces per well). Explants were allowed to adhere to the bottom of the wells and Eagle’s Minimum Essential Medium (EMEM: ATCC; Manassas, VA) containing 10% fetal bovine serum (FBS: ATCC) was added. After 1–2 weeks of cultivation, the cells were passaged into a 100 mm cell culture plate. The cells were allowed to grow to 100% confluence before being used in the culture system. Morphologically, HKCs exhibited characteristic myofibroblastic morphology, such as prominent cytoplasmic actin microfilaments (stress fibers) and high levels of α-smooth muscle actin (SMA) expression (data not shown), that distinguished them from healthy HCFs.

For analysis of variegated, mosaic patterns, it is important to distinguish between different types of patches and clones. We use three terms defined previously [25 ,50 -53 (link)]. A patch is a group of cells of like genotype (or phenotype) that are contiguous at the time of consideration. A descendent clone is any group of clonally related cells irrespective of whether they have remained contiguous throughout development. A coherent clone is a group of clonally-related cells that have remained contiguous throughout development. The number of coherent clones per patch depends partly on the proportions of the two cell populations in the mosaic. For one-dimensional mosaics (strings of two cell populations) the number of coherent clones per patch of cell population 'A' is estimated as 1/(1-p) (where p is the proportion of cell population 'A' in the mosaic) [25 ,28 ,53 (link)].
The observed mean width of β-gal-positive stripes in the corneal epithelium was corrected for the probability that stripes would contain multiple adjacent β-gal-positive corneal epithelial clones. This involved dividing the observed mean width by the function 1/(1-p), where p is the proportion of β-gal-positive cells around the circumference [4 (link),25 ,52 (link),53 (link)]. For radial stripes that form a complete circle, the corrected mean stripe width is identical for β-gal-positive and β-gal-negative stripes because the numbers of β-gal-positive and β-gal-negative stripes are identical and the proportions of β-gal-positive and β-gal-negative cells around the circumference are not independent. The reciprocal of this corrected mean stripe width, expressed as the proportion of the circumference, is the corrected stripe number. This provides an estimate of the total number of corneal epithelial coherent clones (both β-gal-positive and β-gal-negative) per circumference [4 (link),5 (link)]. This estimate assumes (1) coherent clones are equal in size, or their sizes are normally distributed, and (2) β-gal-positive and β-gal-negative coherent clones are randomly distributed around the circumference. Even if these assumptions are not correct the corrected stripe number should be proportional to the number of coherent clones so its application as a comparative measure is still valid.