title: Evidence suggesting the existence of stem cells for the human corneal endothelium.
abstract: OBJECTIVE
  The well-known reluctance of human corneal endothelial cells (HCECs) to divide has continually intrigued investigators. Related to this, the discovery of an increased endothelial cell population in the periphery of the human cornea has prompted an investigation for evidence of the existence of stem-like cells in the endothelial periphery. Showing that stem cells or transient amplifying cells may exist in the periphery might explain the origin of HCECs and indicate a source for these cells in wound repair. In addition, these cells might be of value in culturing or as a source for the synthesis of artificial corneas.
  
  
  METHODS
  Human corneas with attached scleral rims were obtained from eye banks and were assayed for telomerase activity and BrdU (bromodeoxyridine) incorporation to determine, respectively, the presence of a stem-like cell marker and replicative activity. In the case of telomerase activity, the tissues were divided into central, intermediate and peripheral areas by the use of trephines. BrdU staining (using alkaline phosphatase bound secondary antibody) was performed on whole corneas plus scleral rims exposed to BrdU antibodies on the endothelial side whereas BrdU fluorescence (using fluorescein bound secondary antibody) was obtained from transverse sections of the these tissues by the same procedure. Some corneas were wounded to determine whether the wounded areas stimulated BrdU (by staining or fluorescence) followed by the synthesis of transforming growth factor beta (TGFbeta). The latter was determined by quantitative ELISA. Rabbit corneas were also assayed for BrdU incorporation to compare their evidence of cell division with that of humans.
  
  
  RESULTS
  After dividing corneas into central, intermediate, and peripheral sections, the dissected endothelial tissues exhibited positive telomerase activity in the peripheral and intermediate sections. No activity was observed in the central endothelial tissues or the limbus between the trabecular meshwork and Schwalbe's line. BrdU staining with alkaline phosphatase was occasionally observed in the wounded area's human corneal endothelial cells after wounding. When BrdU fluorescence assays were made on corneal transverse sections with fluorescein, fluorescence occurred in an area just at and adjacent to the trabecular meshwork, but was not seen at the corneal endothelium. After wounding, BrdU fluorescence extended into the corneal endothelium. TGF-beta levels were increased in fluids bathing the endothelium following wounding, but the increases lagged behind the wounding event.
  
  
  CONCLUSIONS
  It is suggested stem-like cells may be sequestered in a niche at the junctional region where the corneal endothelial cells and the trabecular meshwork come together. These putative stem cells may supply new cells for both the corneal endothelium and the trabeculae. Evidence suggests that cells from this area migrate (perhaps as transient amplifying cells) to the endothelial periphery and, perhaps, to wounded areas of the corneal endothelium when needed. The migration may not be constant and may be age dependent.
authors.affiliation: Department of Vision Sciences, Vision Science Research Center, School of Optometry, The University of Alabama at Birmingham, 35294, USA. DWhikehart@icare.opt.uab.edu
authors.forename: David R
authors.initials: DR
authors.lastname: Whikehart
authors.forename: Chirag H
authors.initials: CH
authors.lastname: Parikh
authors.forename: Alexia V
authors.initials: AV
authors.lastname: Vaughn
authors.forename: Kathy
authors.initials: K
authors.lastname: Mishler
authors.forename: Henry F
authors.initials: HF
authors.lastname: Edelhauser
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000378:metabolism; D018929:Cell Culture Techniques; D002455:Cell Division / Q000502:physiology; D004247:DNA / Q000096:biosynthesis; D004728:Endothelium, Corneal / Q000166:cytology* / Q000293:injuries / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D009924:Organ Culture Techniques; D011817:Rabbits; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology
chemical_list: D015415:Biomarkers; D016212:Transforming Growth Factor beta; D004247:DNA; D019098:Telomerase; D001973:Bromodeoxyuridine