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S. Yoshida, S. Shimmura, Y. Matsuzaki, J. Shimazaki, H. Okano, K. Tsubota; Multipotent Cornea Stromal Precursors Are of Neural Crest Origin and Not the Bone Marrow . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2979.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the origin of sphere initiating, multipotent corneal stroma–derived presursor cells (COPs).
Cells were dissociated from corneal stroma of adult mice and cultured in sphere forming medium. Methylcellulose culture was used to assess clonal sphere formation from single cells. Cells were cultured in differentiation–inducing medium and differentiation ability was examined by immunocytochemical staining and Oil–Red O staining. Expression of neural stem cell markers was also examined by immunocytochemical analysis and by RT–PCR. To see stem cell specific expression of nestin, COP spheres prepared from E/nestin–EGFP mice. Flow cytometric analysis were performed to examine SP phenotype and expression of surface markers. To investigate the origin of COPs, GFP+ bone marrow cells were transplanted into irradiated B6 mice and cells were prepared from the mice 8 weeks after transplantation. To examine if COPs are originated from neural crest, cells were also prepared from P0–Cre/EGFP mice which express EGFP neural crest–derived cells. Expression of neural crest stem cells–related markers in COPs were examined by RT–PCR.
COPs initiate spheres by clonal expansion from single cells. In addition to dendritic morphology of keratocytes and α–SMA expression, ability to differentiate into adipocytes, chondrocytes, and neural cells was shown by Oil–Red O staining and the expression of collagen II, aggrecan, ß III–tubulin, and NF–M. COPs from E/nestin–EGFP mice showed induction of EGFP expression. COPs were Sca–1+, CD34+, CD45– and c–kit–. Numerous GFP+ cells were observed in the corneas of mice transplanted with GFP+ bone marrow cells, however, no GFP+ COP spheres were initiated from these mice. While, COP spheres from P0–Cre/EGFP were GFP+ indicating the neural crest origin of COPs, which was confirmed by the expression of the embryonic neural crest markers Twist, Snail, Slug, and Sox9.
Sphere initiating cells present in adult mouse corneal stroma were neural crest–derived, multipotent stem cells.
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