Preservation and expansion of the primate keratocyte phenotype by downregulating TGF-beta signaling in a low-calcium, serum-free medium.
OBJECTIVE
To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-beta signaling in rhesus monkey keratocytes expanded in a serum-free and low-[Ca2+] medium.


METHODS
Rhesus monkey keratocytes were isolated from central corneal buttons by collagenase digestion for 16 hours, seeded on plastic in Dulbecco's modified Eagle's medium (DMEM) containing insulin-transferrin-sodium selenite (ITS) supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/10% FBS), or in a defined keratinocyte serum-free medium (KSFM). After confluence, cells in KSFM were continuously subcultured at a 1-to-3 split. Cellular proliferation was analyzed by immunostaining for Ki67 and the MTT assay. The cellular phenotype was determined by immunostaining for aldehyde dehydrogenase (ALDH), keratocan, and CD34 and by the expression of keratocan promoter-driven enhanced cyan fluorescent protein (ECFP). The stability of the keratocyte phenotype was examined by switching KSFM to DMEM/ITS and DMEM/10% FBS. TGF-beta signaling was monitored by measuring the promoter activity of TGF-beta1, -beta2, and -beta RII after transient adenoviral transfection, and cytolocalization of Smad2 and Smad4.


RESULTS
In KSFM, monkey keratocytes proliferated while maintaining the expression of keratocan, CD34, and ALDH proteins and keratocan promoter-driven ECFP for at least 15 passages. The nuclear accumulation of Smad2 and Smad4 and the promoter activities of TGF-beta1 and -beta RII were significantly downregulated in KSFM compared with DMEM/10% FBS. In KSFM, an increase of [Ca2+] to 1.8 mM and addition of 10% FBS synergistically downregulated the keratocan promoter activity, facilitated Smad2 and Smad4 nuclear translocation, and upregulated TGF-beta1 and -beta RII promoter activities.


CONCLUSIONS
The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by downregulating Smad-mediated TGF-beta signaling.
Kawakita
Tetsuya
T
TissueTech, Inc., Ocular Surface Center, Miami, Florida 33176, USA.
Espana
Edgar M
EM
He
Hua
H
Smiddy
Robert
R
Parel
Jean-Marie
JM
Liu
Chia-Yang
CY
Tseng
Scheffer C G
SC
D000256:Adenoviridae / Q000235:genetics; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D049109:Cell Proliferation; D003319:Corneal Stroma / Q000166:cytology* / Q000378:metabolism; D016895:Culture Media, Serum-Free; D015536:Down-Regulation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007124:Immunoenzyme Techniques; D019394:Ki-67 Antigen / Q000378:metabolism; D008253:Macaca mulatta; D051379:Mice; D010641:Phenotype; D011509:Proteoglycans / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology*; D051785:Smad Proteins / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; C099280:Kera protein, mouse; D019394:Ki-67 Antigen; D011509:Proteoglycans; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D000444:Aldehyde Dehydrogenase; D002118:Calcium