Abstract
Purpose: :
Sclera forms the outer coat of the eyeball and provides a strong framework composed by fibrous extracelluar matrix. Cells of sclera secrete specialized extracellular matrix. The aim of this study is to identify scleral stem/progenitor cells (SSPC) derived from murine sclera.
Methods: :
Scleras of C57BL6/J mice were enzyme digested after retina and choroid were removed. Proliferating cells in sclera were studied by clonogenicity, self-renew ablity, proliferation by Brdu and multipotent differentiation capacity. Flow-cytometry, RT-PCR, Western blot analysis and immunofluorescence staining were used for analysis.
Results: :
SSPC cells from mouse scleral tissues appeared spindle fibroblast-like and adherent to the culture plate. SSPCs show abilities of clonal growth, proliferation and self-renewing capacity. These cells showed colony forming ability in 14 days of plating. SSPCs showed a population doubling over 40. Immunophenotyping of these cells by FACS and immunofluorescence showed that the SSPC were positive for mesenchymal markers and negative for hematopoietic markers (Sca-1, CD90.2, CD44, CD105, CD73, CD45-, CD11b-, Flk1-, CD34-, CD117 -). These cells expressed undifferentiated stem cell markers including PAX6, ABCG2, Six2 and NOTCH1 genes, and some lineage-specific markers like alpha-SMA, Vimentin and Collagen type 1. These cells had negative gene expression for epithelium and muscle cell marker like CK12, CK19 and Desmin.The culture of SSPCs can be differentiated toward the adipogenic, chondrogenic, and neurogenic lineages.
Conclusions: :
The results indicate that sclera-derived cells are a new source of multipotent mesenchymal stem cells. These appear to be the first cells from sclera identified with stem/progenitor potential. Further analysis of these cells will aid elucidation of molecular mechanism of sclera development, scleritis and myopia.