Human limbal mesenchymal cells support the growth of human corneal epithelial stem/progenitor cells

Invest Ophthalmol Vis Sci. 2014 Oct 2;55(10):6953-9. doi: 10.1167/iovs.14-14999.

Abstract

Purpose: 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.

Keywords: cornea epithelium; fibroblasts; limbal stem cell deficiency; limbal stem cells; limbus; mesenchymal; stem cell niche; stem cells.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Cell Differentiation
  • Cells, Cultured
  • Epithelium, Corneal / cytology
  • Female
  • Humans
  • Limbus Corneae / cytology*
  • Male
  • Mesenchymal Stem Cells / physiology*
  • Middle Aged
  • Stem Cells / cytology*
  • Young Adult