Abstract
Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it’s presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.
Highlights
Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies
Time-lapse imaging of emerging cultivated limbal epithelial cells (LECs) sheets revealed colonies of corneal epithelial cells forming between feeder cells between days 2 to 4 in culture, followed by corneal epithelial cells proliferating with collective migration (Supplementary Video S1)
There was a tendency for the dynamic behavior of each of the four cultivated LEC sheets to differ
Summary
Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Complete loss of limbal epithelial stem cells due to severe trauma (e.g., thermal and chemical burns) or eye disease (e.g., Stevens-Johnson syndrome or ocular pemphigoid) induces a corneal limbal stem cell deficiency (LCSD)[4,5]. In these situations, the function of renewing the corneal epithelium by generating transient amplifying cells that migrate from the limbus into the corneal basal layer is lost. It is further appreciated that p63, a corneal epithelial stem cell maker, likely influences the proliferation and stratification of epithelial cells in emerging cultivated LEC sheets[15,16,17,18,19]. The purpose of this study was to evaluate p63 and cell jamming in ex vivo explanted cultivated LEC sheets to better understand the dynamics and likely final quality of the generated sheets
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