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(Investigative Ophthalmology and Visual Science. 2004;45:2985-2991.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0201
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CD-34 Expression by Cultured Human Keratocytes Is Downregulated during Myofibroblast Differentiation Induced by TGF-ß1

Edgar M. Espana,1,2 Tetsuya Kawakita,1,2 Chia-Yang Liu,3 and Scheffer C. G. Tseng1,2

1From TissueTech, Inc. and 2Ocular Surface Center, Miami, Florida; and 3Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida.

PURPOSE. To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-ß1–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-ß1 or in DMEM with 1% FBS and 10 ng/mL TGF-ß1. 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-ß1, 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-ß1, 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-ß1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-ß signaling.




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