CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-β1

Edgar M. Espana, Tetsuya Kawakita, Chia Yang Liu, Scheffer C G Tseng

Research output: Contribution to journalArticle

59 Citations (Scopus)

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 languageEnglish
Pages (from-to)2985-2991
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number9
DOIs
Publication statusPublished - 2004 Sep
Externally publishedYes

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Amnion
Myofibroblasts
Plastics
Down-Regulation
Serum-Free Culture Media
Serum
Sodium Selenite
Collagenases
Transferrin
Cornea
Smooth Muscle
Actins
Western Blotting
Maintenance
Insulin

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 journalArticle

Espana, Edgar M. ; Kawakita, Tetsuya ; Liu, Chia Yang ; Tseng, Scheffer C G. / CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-β1. In: Investigative Ophthalmology and Visual Science. 2004 ; Vol. 45, No. 9. pp. 2985-2991.
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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.",
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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

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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.

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