title: Human limbal mesenchymal cells support the growth of human corneal epithelial stem/progenitor cells.
abstract: OBJECTIVE
  We tested the viability of human limbal mesenchymal cells (LMCs) to support the expansion of human corneal epithelial stem/progenitor cells (LSCs).
  
  
  METHODS
  Human LMCs were isolated from sclerocorneal tissue using collagenase A. Primary limbal epithelial cells (LECs) in the form of single cell suspension or cell clusters were cocultured on a monolayer of either 3T3 cells (control) or LMCs (SC-LMC culture). The LEC clusters also were grown directly on LMCs (CC-LMC culture) and in an optimized 3-dimensional culture method (3D CC-LMC culture). Colony-forming efficiency (CFE) and LEC proliferation were analyzed. The phenotype of the cultured LECs was assessed by their expression level of putative stem cell markers and a differentiation marker by qRT-PCR and immunocytochemistry.
  
  
  RESULTS
  The LECs in the SC-LMC culture had a very limited growth and the stem/progenitor phenotype was lost compared to the control. Growth and cell morphology improved using the CC-LMC culture. The 3D CC-LMC culture method was the best to support the growth of the LSC population. Expression of ATP-binding cassette family G2 and ΔNp63 at the mRNA level was maintained or increased in CC-LMCs and 3D CC-LMC cultures compared to the control. The percentage of the K14(+) and K12(+) cells was comparable in these three cultures. There was no significant difference in the percentage of p63α high expressing cells in the control (21%) and 3D CC-LMC culture (17%, P > 0.05).
  
  
  CONCLUSIONS
  Human LMCs can substitute 3T3 cells in the expansion of LSCs using the 3-dimensional culture system.
authors.affiliation: Cornea Division, Jules Stein Eye Institute, University of California, Los Angeles, California, United States.
authors.forename: Martin N
authors.initials: MN
authors.lastname: Nakatsu
authors.affiliation: Cornea Division, Jules Stein Eye Institute, University of California, Los Angeles, California, United States.
authors.forename: Sheyla
authors.initials: S
authors.lastname: González
authors.affiliation: Cornea Division, Jules Stein Eye Institute, University of California, Los Angeles, California, United States.
authors.forename: Hua
authors.initials: H
authors.lastname: Mei
authors.affiliation: Cornea Division, Jules Stein Eye Institute, University of California, Los Angeles, California, United States.
authors.forename: Sophie X
authors.initials: SX
authors.lastname: Deng
mesh_terms: D000328:Adult; D000368:Aged; D002454:Cell Differentiation; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000166:cytology; D005260:Female; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D008875:Middle Aged; D013234:Stem Cells / Q000166:cytology*; D055815:Young Adult
keywords: cornea epithelium; fibroblasts; limbal stem cell deficiency; limbal stem cells; limbus; mesenchymal; stem cell niche; stem cells