Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 2985-2991 |
| Number of pages | 7 |
| Journal | Investigative Ophthalmology and Visual Science |
| Volume | 45 |
| Issue number | 9 |
| DOIs | |
| Publication status | Published - 2004 Sep |
| Externally published | Yes |
Fingerprint
ASJC Scopus subject areas
- Ophthalmology
Cite this
CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-β1. / Espana, Edgar M.; Kawakita, Tetsuya; Liu, Chia Yang; Tseng, Scheffer C G.
In: Investigative Ophthalmology and Visual Science, Vol. 45, No. 9, 09.2004, p. 2985-2991.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-β1
AU - Espana, Edgar M.
AU - Kawakita, Tetsuya
AU - Liu, Chia Yang
AU - Tseng, Scheffer C G
PY - 2004/9
Y1 - 2004/9
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=4344627864&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4344627864&partnerID=8YFLogxK
U2 - 10.1167/iovs.04-0201
DO - 10.1167/iovs.04-0201
M3 - Article
C2 - 15326111
AN - SCOPUS:4344627864
VL - 45
SP - 2985
EP - 2991
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 9
ER -