PURPOSE. To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-β1-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-β1 or in DMEM with 1% FBS and 10 ng/mL TGF-β1. Protein expression of CD34 and α-smooth muscle actin (α-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 α-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed α-SMA without CD34 when continuously passaged on plastic. Expression of α-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas α-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-β1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became α-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-β1, 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-β1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-β signaling.
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience