CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-beta1.
OBJECTIVE
To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-beta1-induced myofibroblast differentiation.


METHODS
Collagenase-isolated keratocytes were seeded and subcultured on plastic or amniotic membrane matrix (AM) in DMEM, with or without 10% FBS, in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS) with 10, 100, and 1000 pg/mL TGF-beta1 or in DMEM with 1% FBS and 10 ng/mL TGF-beta1. Protein expression of CD34 and alpha-smooth muscle actin (alpha-SMA) was measured by Western blot and immunostaining.


RESULTS
Keratocytes, expressing CD34 in normal human corneas, continued to express CD34 when cultured on AM in serum-containing medium and on plastic in serum-free medium, but expression was lost on plastic in serum-containing medium. In serum-containing medium, expression of CD34, but not alpha-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed alpha-SMA without CD34 when continuously passaged on plastic. Expression of alpha-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas alpha-SMA expression was upregulated when cells were subcultured on plastic. In serum-free medium, CD34 expression was maintained by cells treated with 10 pg/mL TGF-beta1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became alpha-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-beta1, but failed to do so if cultured on AM, even for 7 days.


CONCLUSIONS
These findings indicate that CD34 is expressed by human keratocytes in vivo and in vitro. Myofibroblast differentiation promoted by TGF-beta1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-beta signaling.
Espana
Edgar M
EM
TissueTech, Inc., Miami, Florida 33173, USA.
Kawakita
Tetsuya
T
Liu
Chia-Yang
CY
Tseng
Scheffer C G
SC
D000199:Actins / Q000378:metabolism; D000650:Amnion / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology* / Q000378:metabolism*; D015536:Down-Regulation; D005347:Fibroblasts / Q000166:cytology*; D006801:Humans; D007158:Immunologic Techniques; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D000199:Actins; D018952:Antigens, CD34; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1