TY - JOUR
T1 - Preservation and expansion of the primate keratocyte phenotype by downregulating TGF-β signaling in a low-calcium, serum-free medium
AU - Kawakita, Tetsuya
AU - Espana, Edgar M.
AU - He, Hua
AU - Smiddy, Robert
AU - Parel, Jean Marie
AU - Liu, Chia Yang
AU - Tseng, Scheffer C.G.
PY - 2006/5
Y1 - 2006/5
N2 - PURPOSE. To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-β 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-β signaling was monitored by measuring the promoter activity of TGF-β1, -β2, and -β 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-β1 and -β RII were significantly down-regulated 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-β1 and -β RII promoter activities. CONCLUSIONS. The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by down-regulating Smad-mediated TGF-β signaling.
AB - PURPOSE. To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-β 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-β signaling was monitored by measuring the promoter activity of TGF-β1, -β2, and -β 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-β1 and -β RII were significantly down-regulated 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-β1 and -β RII promoter activities. CONCLUSIONS. The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by down-regulating Smad-mediated TGF-β signaling.
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U2 - 10.1167/iovs.05-1040
DO - 10.1167/iovs.05-1040
M3 - Article
C2 - 16638999
AN - SCOPUS:33744751473
VL - 47
SP - 1918
EP - 1927
JO - Investigative Ophthalmology
JF - Investigative Ophthalmology
SN - 0146-0404
IS - 5
ER -