title: Mesenchymal stem cells derived from human limbal niche cells.
abstract: OBJECTIVE
  We investigated whether human limbal niche cells generate mesenchymal stem cells.
  
  
  METHODS
  Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells.
  
  
  RESULTS
  Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α.
  
  
  CONCLUSIONS
  In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.
authors: Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.
authors: Gui-Gang
authors: GG
authors: Li
authors: Ying-Ting
authors: YT
authors: Zhu
authors: Hua-Tao
authors: HT
authors: Xie
authors: Szu-Yu
authors: SY
authors: Chen
authors: Scheffer C G
authors: SC
authors: Tseng
mesh_terms: D000293:Adolescent; D000328:Adult; D001672:Biocompatible Materials; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003094:Collagen; D003319:Corneal Stroma / Q000166:cytology; D004338:Drug Combinations; D019573:Epithelium, Corneal / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007797:Laminin; D016850:Limbus Corneae / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D008875:Middle Aged; D018919:Neovascularization, Physiologic / Q000502:physiology*; D020286:Pericytes / Q000166:cytology / Q000378:metabolism; D010969:Plastics; D011509:Proteoglycans; D012038:Regeneration / Q000502:physiology; D055153:Stem Cell Niche / Q000502:physiology*; D014945:Wound Healing / Q000502:physiology*; D055815:Young Adult
chemical_list: D001672:Biocompatible Materials; D015415:Biomarkers; D004338:Drug Combinations; D007797:Laminin; D010969:Plastics; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen