The heterogeneous murine corneal stromal cell populations in vitro.
OBJECTIVE
To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin.


METHODS
Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined.


RESULTS
Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1.


CONCLUSIONS
Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.
Espana
Edgar M
EM
TissueTech, Inc. and Ocular Surface Center, Miami, Florida 33173, USA.
Kawakita
Tetsuya
T
Di Pascuale
Mario A
MA
Li
Wei
W
Yeh
Lung-Kun
LK
Parel
Jean-Marie
JM
Liu
Chia-Yang
CY
Tseng
Scheffer C G
SC
D000199:Actins / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008858:Microscopy, Phase-Contrast; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D010641:Phenotype; D011509:Proteoglycans / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014746:Vimentin / Q000378:metabolism
D000199:Actins; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; C099280:Kera protein, mouse; C577484:NES protein, human; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D011509:Proteoglycans; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D014746:Vimentin; D017493:Leukocyte Common Antigens