title: Treatment of rabbit bullous keratopathy with precursors derived from cultured human corneal endothelium.
abstract: OBJECTIVE
  To establish a method for the mass production of human corneal endothelium (HCE) precursors and the therapeutic application of these cells in a rabbit CE-deficiency model.
  
  
  METHODS
  A sphere-forming assay was performed to produce precursors from cultured HCE. Various marker expressions were examined in the sphere colonies, and their progenies by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The transport activity of the sphere-derived cell sheet was evaluated by the Ussing chamber system. DiI-labeled precursors obtained from cultured HCE were injected the anterior chamber of the eye in a rabbit CE-deficiency model, and the eye-down position was maintained for 24 hours for attachment to Descemet's membrane (sphere eye-down group). The sphere eye-down and control groups, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations.
  
  
  RESULTS
  Cultured HCE formed primary and secondary sphere colonies. The spheres expressed alpha-smooth muscle actin and nestin, and progeny expressed alpha-smooth muscle actin, confirmed by RT-PCR. The progeny showed an HCE-like hexagonal shape, were confluent, and had adequate transport activity. Mean corneal thickness in the sphere eye-down group was significantly less than in the other control groups 14, 21, and 28 days (P < 0.006) after surgery. The HCE-like hexagonal cells detected on the Descemet's membrane are DiI-positive in the sphere eye-down group.
  
  
  CONCLUSIONS
  The findings demonstrate that culture of HCE can promote mass production of HCE precursors, determined by sphere-forming assay. Injection of precursors derived from cultured HCE into the anterior chamber is an effective treatment strategy for CE deficiency in a rabbit model.
authors.affiliation: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Mimura
authors.forename: Seiichi
authors.initials: S
authors.lastname: Yokoo
authors.forename: Makoto
authors.initials: M
authors.lastname: Araie
authors.forename: Shiro
authors.initials: S
authors.lastname: Amano
authors.forename: Satoru
authors.initials: S
authors.lastname: Yamagami
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000328:Adult; D000818:Animals; D000867:Anterior Chamber; D002465:Cell Movement; D049109:Cell Proliferation; D003316:Corneal Diseases / Q000473:pathology / Q000601:surgery*; D004195:Disease Models, Animal; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D064231:Nestin; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018874:Spheroids, Cellular / Q000166:cytology; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D000199:Actins; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin