PURPOSE: To identify adult human buccal epithelial stem cells (SCs) on the basis of two parameters (high p63 expression and greater nucleus/cytoplasmic (N/C) ratio) and to evaluate clinical efficacy of ex-vivo expanded autologous epithelium in bilateral limbal SC-deficient (LSCD) patients. METHODS: The epithelial cells were isolated from buccal biopsy and cultured on human amnion in culture inserts with 3T3 feeder layer. The SCs were identified on the basis of two-parameter analysis using confocal microscopy, surface markers, and colony-forming efficiency (CFE). The cultured epithelium was transplanted in 10 LSCD patients followed by penetrating keratoplasty in 4 patients. The clinical outcome was followed up to 3 years. RESULTS: A distinct population (3.0±1.7%) of small cells expressing high levels of p63 with greater N/C ratio was observed in buccal epithelium. The N/C ratio was found to be more appropriate than cell diameter for two-parameter analysis. These cells located in the basal layer were negative for connexin-43 and positive for melanoma-associated chondroitin sulfate proteoglycan, containing holoclones with 0.2% CFE, thus representing the SC population. After transplantation of cultured epithelium with increased (sixfold) SC content, anatomical and visual improvement was observed at 13-34 months in 3/10 LSCD patients. CONCLUSIONS: The two-parameter SC marker is useful to identify and quantify buccal epithelial SCs. The transplantation of bioengineered SC-rich buccal epithelium is a strategy for corneal surface reconstruction in bilateral LSCD. However, further studies are required to optimize the culture conditions and to look for other sources of adult SCs for better visual outcome.
PURPOSE: To identify adult human buccal epithelial stem cells (SCs) on the basis of two parameters (high p63 expression and greater nucleus/cytoplasmic (N/C) ratio) and to evaluate clinical efficacy of ex-vivo expanded autologous epithelium in bilateral limbal SC-deficient (LSCD) patients. METHODS: The epithelial cells were isolated from buccal biopsy and cultured on human amnion in culture inserts with 3T3 feeder layer. The SCs were identified on the basis of two-parameter analysis using confocal microscopy, surface markers, and colony-forming efficiency (CFE). The cultured epithelium was transplanted in 10 LSCD patients followed by penetrating keratoplasty in 4 patients. The clinical outcome was followed up to 3 years. RESULTS: A distinct population (3.0±1.7%) of small cells expressing high levels of p63 with greater N/C ratio was observed in buccal epithelium. The N/C ratio was found to be more appropriate than cell diameter for two-parameter analysis. These cells located in the basal layer were negative for connexin-43 and positive for melanoma-associated chondroitin sulfate proteoglycan, containing holoclones with 0.2% CFE, thus representing the SC population. After transplantation of cultured epithelium with increased (sixfold) SC content, anatomical and visual improvement was observed at 13-34 months in 3/10 LSCD patients. CONCLUSIONS: The two-parameter SC marker is useful to identify and quantify buccal epithelial SCs. The transplantation of bioengineered SC-rich buccal epithelium is a strategy for corneal surface reconstruction in bilateral LSCD. However, further studies are required to optimize the culture conditions and to look for other sources of adult SCs for better visual outcome.
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