Kinetics of expansion of human limbal epithelial progenitor cells in primary culture of explants without feeders

Abstract

The aims of this study were to determine whether human limbal explant cultures without feeder cells result in expansion of epithelial progenitors and to estimate the optimal expansion time for progenitor cells. Limbal explants from ten human corneas were cultured for 7, 9, 11, 14, 18, and 21 days. Limbal explants from two corneas were enzymatically dissociated or directly cultured for 14 days. Progenitor cells were characterized by their ability to form colonies, by immunocytochemistry, and by quantitative real-time polymerase chain reaction. Colonies were identified after 9, 11, 14, and 18 days of culture, but not after 21 days. The number of colonies per explant was significantly higher after 14 days than after 9 and 21 days. The mean percentage of seeded cells giving rise to clones was 4.03% after 14 days of culture and 0.36% for non-cultured dissociated limbal epithelial cells. The number of cells giving rise to clones per cornea significantly increased from an average of 2275 for non-cultured cells to 24266 for cells cultured for 14 days. Immunocytochemical analysis detected positive staining for cytokeratin (CK) 3, CK5/6/8/10/13/18, CK19, vimentin, p63, and p63α, in both cultures and clones. CK3 expression increased significantly with culture time. Transcript expression was observed for CK3, CK19, vimentin, and Delta N p63α at each culture time point, both in cultures and clones. The optimal culture time for limbal explants in cholera toxin-free Green medium without feeder cells was 14 days leading to the expansion of progenitors.

Figure 1. Experimental design.

A. First series of experiments: 60 human superficial limbal explants were sutured on a tissue-culture treated round coverslip epithelium side up and cultured for 7 to 21 days using 6-well plates. B. Second series of experiments: 6 superficial limbal explants from two human donor corneas (3 per cornea) were sutured and cultured for 14 days and the remaining 6 explants were digested with collagenase A.

Figure 2. Image analysis of stained cultured cells.

An image analysis algorithm was developed to detect individual cell contours and to measure the level of brown coloration using ImageJ. A. Primary Image. B. Mask of cells obtained from the green channel of the primary image (Threshold followed by watershed). C. Inverted mask of cells redirected to the primary image. D. Blue channel of the primary image permitting the brown coloration level to be determined.

Figure 3. Number of cells after human limbal explant culture.

Ten limbal rims retrieved from 10 human corneas were divided in 6 explants each and cultured for 7, 9, 11, 14, 18, or 21 days. Only trypan blue negative cells grown out of the explants were counted.

Figure 4. Clonal growth on a 3T3 feeder cell layer and colony-forming efficiency.

A Macroscopic appearance of colonies obtained from cells grown from explants cultured for 9, 11, 14, 18, and 21 days and stained with crystal violet. B Photograph of microscopic appearance of limbal stem cell (LSC) colonies. At the end of primary culture, cells were dissociated and cultured with mitomycin-arrested 3T3 murine feeder cells for 12 days (2 cultures for each primary culture). Colonies were assessed at the end of culture. C Significantly higher CFE (%) was obtained after 9, 11, and 14 days of primary culture compared with 21 days (p=0.00002; ANOVA). Shown are mean + standard errors of the mean and post-hoc tests. D The primary culture time significantly influenced the CFE (N) (number of cells obtained at the end of primary culture * number of clones / number of seeded cells). Significantly higher CFE (N) was obtained after 14 days of primary culture compared with 9 and 21 days. (p=0.004; ANOVA). Shown are mean + standard errors of the mean and post-hoc tests.

Figure 5. Percentage of CK3-expressing cells after primary culture of human limbal explants for 9 to 21 days.

CK3 expression increased with culture time from day 9 to day 21 (p= 0.009).

Figure 6. Comparison of dissociated limbal epithelial cells with cultured limbal epithelial cells for 14 days.

A Colony forming efficiency expressed as the percentage of seeded cells giving rise to clone (CFE %). Significantly higher CFE (%) (p=0.000002) was obtained after 14 days of primary culture of limbal explants compared with non-cultured dissociated limbal epithelial cells. B, C Immunohistochemical analysis of enzymatically dissociated cells and cells cultured for 14 days from human limbal explants. B Primary culture: expression of differentiation markers and progenitors markers was significantly higher in cells cultured for 14 days than in dissociated cells from the explants (p < 0.02) except for CK3 (p = 0.44). C Clones: immunostaining of differentiation and progenitor markers was significantly higher in colonies obtained from cells cultured for 14 days than in colonies obtained from cells dissociated from explants (p < 0.02). Shown are mean + standard errors of the mean.

Figure 7. Colony-forming efficiency (N) expressed as the number of cells giving rise to clones per cornea.

The number of cells giving rise to clones increased from an average of 2275 per cornea for non-cultured cells to 24266 per cornea for cells obtained after culture of limbal explants for 14 days and decreased afterwards (p<0.00001). This figure was close to 0 after 21 days of primary culture. Shown are mean + standard errors of the mean and post-hoc tests.