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1From TissueTech, Inc. and 2Ocular Surface Center, Miami, Florida; and 3Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida.
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Abstract |
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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 
-smooth muscle actin (-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 -SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed -SMA without CD34 when continuously passaged on plastic. Expression of -SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas -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 -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.
-smooth muscle actin (-SMA)–expressing myofibroblasts.5 6
This transition from keratocytes into scar-producing myofibroblasts can be reproduced in vitro. On plastic, bovine,7 and rabbit8 9 keratocytes maintain a dendritic morphology if cultured in serum-free medium. Under such a condition, they express keratan sulfate-containing proteoglycans over dermatan sulfate-containing proteoglycans.10 11 However, when serum is added, bovine keratocytes rapidly lose their dendritic morphology and acquire a fibroblastic morphology.7 If keratocytes are cultured at low densities12 or stimulated by TGF-ß1 in 1% serum or serum-free conditions, they differentiate into myofibroblasts with a spread morphology and prominent focal adhesions, express -SMA, and upregulate integrin 5ß1, cadherins, collagen type I and III, biglycan, and the EDA (EIIIA) form of fibronectin.9 11 13 14 Recently, we have reported that, unlike plastic cultures, human keratocytes grown on amniotic membrane (AM) stromal matrix maintain their dendritic morphology and keratocan expression, even in serum-containing medium for up to five passages.15
CD34, a 110-kDa glycosylated transmembrane protein, has been used as a hematopoietic stem cell marker16 17 and for isolating hematopoietic stem cells pursuant to its transmembrane location. Recently, expression of CD34 has been found in keratocytes in human corneas,18 19 and its expression is lost in several corneal diseases with or without stromal scarring.18 Herein, we provide experimental evidence to support the notion that CD34 can be used as a membrane marker for human keratocytes and is continuously expressed by keratocytes maintained in serum-free medium or expanded by serum on AM cultures, but is lost during myofibroblast differentiation stimulated by TGF-ß1. On AM, keratocytes continued to express CD34 without myofibroblast differentiation, even if challenged with a high dose of TGF-ß1. The significance of these findings is further discussed.
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Materials and Methods |
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-SMA were from Dako (Carpinteria, CA). Collagenase A was obtained from Roche (Indianapolis, IN). All other materials such as minobenzamidine, chloroform EDTA tetrasodium salt, guanidine, human recombinant TGF-ß1, hydrochloric acid, isopropanolol, insulin-transferrin-sodium selenite (ITS), sodium acetate, urea, and mouse monoclonal antibodies against -SMA and ß-actin were from Sigma-Aldrich (St. Louis, MO).
Isolation and Culturing of Keratocytes on Plastic or Amniotic Membrane
Human corneas stored in humid chambers less than 4 days old were obtained from the Florida Lions Eye Bank. Keratocytes were isolated by collagenase, as recently reported.15 In short, the corneal button, removed by an 8.25-mm Barron’s trephine, was incubated at 37°C in DMEM containing 20 mM HEPES and 1.25 mg/mL collagenase A. After 2 to 3 hours of incubation, an already loose corneal epithelium was removed with a cell scraper, and the remaining corneal stroma was cut into four symmetrical pieces and continuously digested overnight at 37°C on a noncoated plastic dish until the tissue became "smeared" onto the dish bottom. The digested tissue was pipetted three times and centrifuged at 800g for 5 minutes and finally resuspended in either 1.5 mL of DMEM containing 10% FBS, 20 mM HEPES, 50 µg/mL gentamicin, and 1.25 µg/mL amphotericin or 1.5 mL DMEM containing 20 mM HEPES and ITS (5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL sodium selenite) per cornea. This keratocyte-containing cell suspension was then seeded on plastic dishes or on the stromal side of denuded AM obtained from Bio-Tissue (Miami, FL), as previously described.15 Cells were cultured in DMEM containing 10% FBS, 20 mM HEPES, 50 µg/mL gentamicin, and 1.25 µg/mL amphotericin (DMEM/10%FBS), or in serum-free DMEM containing 20 mM HEPES and ITS (DMEM/ITS).
Cells cultured on AM were trypsinized near confluence and subcultured at 1:2 split onto either AM (abbreviated as AA) or to plastic (as AP). In parallel, cells expanded on plastic dishes were similarly trypsinized and subcultured at 1:2 split onto plastic (PP) or on AM (PA). Near confluence, cells of AA and AP were subcultured in a similar fashion onto AM stroma (AAA and APA, respectively), whereas cells of PP and PA were subcultured on plastic (PPP and PAP, respectively).
TGF-ß1 Challenge
To test whether exogenous addition of TGF-ß1 would alter CD34 expression during myofibroblast differentiation, defined by -SMA expression, an equal number of collagenase-isolated keratocytes (
1500–2000 cells per cm2) were seeded on plastic for 1 day in DMEM/ITS, and challenged with 10, 100, or 1000 pg/mL of TGF-ß1 for 5 days. In parallel, cells were seeded on AM for 1 day in the same medium and challenged with 1 ng/mL TGF-ß1 for 5 days. To test the inhibitory effect of AM stroma on the expression of -SMA after TGF-ß1 challenge, primary culture of keratocytes expanded on AM were passaged at 1:2 split to plastic and AM, respectively, in DMEM/10% FBS. At 80% confluence, both cultures were switched to DMEM containing 1% FBS and stimulated with10 ng/mL TGF-ß1 for 7 days.
Immunostaining
Normal human corneas obtained from the Florida Lions Eye Bank were embedded in optimal cutting temperature (OCT; Sakura Finetek, Torrance, CA) compound and snap frozen in liquid nitrogen. All sections were fixed in cold acetone for 10 minutes at –20°C and blocked and permeabilized as previously described.20 Cells cultured on plastic or AM were washed three times with PBS after the culture medium was removed and then fixed in the dishes with cold methanol (–20°C) for 5 minutes. After the reaction was blocked with 1% BSA for 30 minutes, cells were incubated for 1 hour with antibody against CD34 (1:40 dilution). Specific binding was detected by a FITC-conjugated anti-mouse secondary antibody (1:100 dilution), counterstained with propidium iodine, and mounted in antifade solution (Vector Laboratories, Burlingame, CA). Primary antibodies were also detected with an immunoperoxidase protocol (ABC kit, Vectastain Elite; Vector Laboratories) and developed with a 3,3'-diaminobenzidine (DAB; Dako). Images were photographed with an epifluorescence microscope (Te-2000u Eclipse; Nikon, Tokyo, Japan).
Western Blot Analysis
The central corneal button was obtained by an 8.25-mm trephine from a normal cornea less than 3 days after harvesting. After the corneal epithelium was removed with a cell scraper and peeling off the endothelium, the remaining stroma was minced with a blade and homogenized at 6000 rpm in 500 µL of radioimmunoprecipitation (RIPA) extraction buffer with a homogenizer (Tissue Tearor; BioSpec, Bartlesville, OK). Cultured cells on AM or plastic dishes were scraped and similarly extracted with RIPA buffer. Proteins were loaded in equal volumes according to a ß-actin loading control. An equal volume of 2x SDS sample buffer was added to samples, boiled for 5 minutes, electrophoresed on an SDS-PAGE gradient (4%–15%) gel, and transferred to a nitrocellulose membrane. These membranes were preincubated with blocking buffer (5% fat free milk) and probed with monoclonal antibodies to CD34 (1:300 dilution), -SMA (1:1000), and ß-actin (1:3000). Immunoreactivity was visualized with a chemiluminescence reagent (Perkin Elmer, Boston, MA).
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Results |
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110 kDa consistent with reported CD3419 and one unknown minor band with a smaller molecular weight (Fig. 1C) . RT-PCR detected CD34 from mRNAs extracted from human corneal stroma but not epithelium (data not shown). These results indicate that CD34 is expressed by keratocytes, but not by the epithelium and endothelium, of human corneas in vivo.
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-SMA was not expressed by the former two conditions, but was expressed by the latter one (Fig. 3A) . These results indicate that expression of CD34 is not compatible with that of -SMA. Expression of CD34 was maintained in culturing conditions known to maintain the keratocyte phenotype, whereas expression of -SMA was consistent with the culturing condition promoting myofibroblast differentiation.
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-SMA Expression during Continuous Subpassages-SMA could be modulated by continuous passages between plastic and AM, protein extracts after the second and third passages were analyzed by Western blot analysis (Fig. 3B) . The results showed that expression of CD34 could be detected only in cells continuously subcultured on AM (i.e., AA and AAA), under which condition there was no expression of -SMA. In contrast, expression of CD34 was lost in cells continuously subcultured on plastic (i.e., PP and PPP), under which condition there was abundant expression of -SMA. Cells expressing -SMA on plastic cultures did not reexpress CD34 when subcultured on AM (i.e., PA and APA), under which condition their prior expression of -SMA (also see Fig. 3A ) was abolished. Cells expressing CD34 on AM cultures express -SMA when subcultured on plastic (i.e., AP and PAP), under which condition their prior expression of CD34 was also abolished.
Immunostaining confirmed the above Western blot analysis data and further showed that some, but not all, cells turned into myofibroblasts with prominent stress fibers in the cytoplasm and expressed -SMA when subcultured to plastic either from AM or plastic at the second passage (i.e., AP and PP, respectively; Fig. 4A and 4B , respectively) and at the third passage (i.e., PAP and PPP, respectively; Fig. 4C and 4D , respectively). In contrast, cells cultured on AM did not expressed -SMA, no matter if they were subcultured from AM or plastic, so long as they were grown on AM at the second passage (i.e., AA and PA, respectively; not shown) and at the third passage (i.e., AAA and APA, respectively; Figs. 4E 4F , respectively). These findings indicate that expression of CD34 and -SMA was differently modulated. That is, upregulation of CD34 was associated with downregulation of -SMA, whereas upregulation of -SMA was associated with downregulation of CD34. Such different modulation of CD34 and -SMA expression could be manipulated by continuous subculturing between plastic and AM. Subculturing cells from AM to plastic lost CD34 expression and promoted -SMA expression. Subculturing cells from plastic to AM, however, suppressed -SMA expression, but did not regain expression of CD34.
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-SMA Expression-SMA (Figs. 5C) . At 10 pg/mL TGF-ß1, cells remained dendritic and uniformly spread (Fig. 5D) and expressed CD34 weakly (Fig. 5E) , but not -SMA (Fig. 5F) . At 100 pg/mL TGF-ß1, cells became spindle shaped and started aggregation (Fig. 5G) . Notably, cells lost CD34 expression (Fig. 5H) , but showed positive -SMA expression (Fig. 5I) . At 1 ng/mL TGF-ß1, cells became spindle-shaped and aggregated into clusters (Fig. 5J) . They lost CD34 expression (Fig. 5K) , and strongly expressed -SMA, especially in cell aggregates (Fig. 5L) . In contrast, cells cultured on AM at 1 ng/mL TGF-ß1, maintained a dendritic morphology without aggregation (Fig. 5M) and expressed CD34 (Fig. 5N) . No -SMA expression was observed (Fig. 5O) .
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-SMA staining in cytoplasmic stress fibers of nearly all cells cultured on plastic (Fig. 6A) . In contrast, all cells cultured on AM remained negative to -SMA staining (Fig. 6B) . Western blot analysis confirmed that -SMA was expressed by cells on plastic after the treatment of 10 ng/mL TGF-ß1, but not by cells on AM treated with or without 10 ng/mL TGF-ß1 (Fig. 6C) . Collectively, these results showed that TGF-ß1 was a potent inducer of myofibroblast differentiation for cells cultured on plastic, especially in serum-free medium or medium with 1% FBS. Such an induction was blocked when cells were cultured on AM.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-SMA-expressing myofibroblasts on plastic under the influence of serum (Fig. 3A) , a condition also known to stop keratocan expression.15 These data also indicate that CD34 may be used as a membrane marker to isolate keratocytes for additional manipulations in the future.
Not only in primary cultures, expression of CD34 was modulated differently from that of -SMA during subsequent passages in DMEM/10% FBS. CD34 was expressed by cells continuously subcultured on AM, under which condition there was no expression of -SMA (Fig. 3B) . In contrast, expression of CD34 was lost in cells continuously subcultured on plastic, under which condition -SMA was expressed. These data strongly confirm that upregulation of CD34 is associated with downregulation of -SMA, whereas upregulation of -SMA is associated with downregulation of CD34. Furthermore, subculturing cells from AM to plastic lost CD34 expression and promoted -SMA expression (Fig. 3B) . Subculturing cells from plastic to AM suppressed -SMA expression, but did not regain expression of CD34. Previously, we have reported that cells subcultured from plastic to AM also do not regain expression of keratocan.15 CD34 expression is lost in a number of corneal diseases with or without histopathological evidence of scarring.18 We thus speculate that cells not expressing CD34 or -SMA may represent a "transitional fibroblast state," between keratocytes and -SMA–expressing myofibroblasts, a concept that has been proposed by others.9 11 Experimental subculturing of cells from plastic to AM may offer a unique opportunity to test our hypothesis.
It should be noted that not all cells that lost CD34 expression turned into -SMA–expressing myofibroblasts when cultured on plastic in the presence of serum (Fig. 4) . Serum is known to contain both agonists and antagonists in promoting myofibroblast differentiation.8 24 25 26 To circumvent this problem, the primary culture was switched to serum-free DMEM/ITS and treated with increasing concentrations of TGF-ß1 from 10 to 1000 pg/mL. Several studies have documented the profibrotic action of TGF-ß1 in corneal keratocytes.8 27 In vivo, addition of a monoclonal antibody that recognizes TGF-ß1, -ß2, and -ß3 blocks its fibrotic response in rabbit corneas after lamellar keratectomy28 and extensive PRK.29 The profibrotic action of TGF-ß1 is also accompanied by the loss of the keratocyte phenotype. As stated in the introduction, primary cultures of bovine keratocytes in DMEM/F12 with 2% FBS transform into myofibroblasts in response to exogenous 2 ng/mL TGF- ß1.11 Myofibroblasts characteristically downregulated keratan sulfate synthesis and increased biglycan secretion while upregulating fibronectin and -SMA.11 Similarly, rabbit keratocytes during primary culture in DMEM did not express -SMA, whereas the same cells became myofibroblastic when treated with 1 ng/mL TGF-ß1 as evidenced by the spread morphology, F-actin filament bundles, and expression of fibronectin and -SMA.9 In this study, we noted that although downregulated compared with the control, CD34 was still expressed by cells treated with TGF-ß1 as low as 10 pg/mL (Fig. 5E) . This situation resembles what has been reported for human hematopoietic cells where CD34 expression is found in hematopoietic stem cells, immature myeloid cell lines, and leukemic marrow cells but decreases during differentiation.30 Future studies are needed to determine whether indeed some CD34-positive keratocytes are less differentiated than has been thought to be the case so far.
Nevertheless, when TGF-ß1 was added, more than 100 pg/mL, progressive morphologic changes were induced from dendritic to fibroblastic with notable cell aggregation (Fig. 5) . Furthermore, such morphologic changes were accompanied by downregulation of CD34 and upregulation of -SMA in nearly all cells on plastic cultures. These results indicated that TGF-ß1 is a potent cytokine inducing keratocytes to lose their characteristic expression of CD34 and to become myofibroblasts.
A striking finding was that both fibroblastic morphology and expression of -SMA, induced by 1 ng/mL of TGF-ß1 in DMEM/ITS and by 10 ng/mL of TGF-ß1 in DMEM/1% FBS for 5 and 7 days, respectively, were inhibited when cells were cultured on AM (Fig. 6) . Again, suppression of -SMA-expressing myofibroblast phenotype was correlated with preservation of CD34-expressing keratocytes. These results, collectively, support our prior speculation that cells cultured on AM withstand TGF-ß1 signaling. Indeed, we have reported that transcript expression of TGF-ß2, ß3, and -ßRII transcripts by human corneal and limbal fibroblasts is suppressed when cells are cultured on AM in serum-containing or serum-free medium, of which the latter is challenged by exogenous 10 ng/mL TGF-ß1,31 and that transcript expression of TGF-ß2, -ß3, -ßRI, -ßRII, and -ßRIII by human conjunctival and pterygium fibroblasts is also suppressed when cells are cultured on AM in serum-containing or -free medium, of which the latter is challenged by exogenous 10 ng/mL TGF-ß1.32 Nevertheless, transcript expression of these genes is maintained and further stimulated by exogenous TGF-ß1 when cells were cultured on plastic.31 32 Therefore, we believe that suppression of TGF-ßRI, -II, and -III and TGF-ß1 and -ß2 at the transcriptional level is potent enough to turn off signaling mediated by exogenous TGF-ß1 when cells are cultured on AM. Studies are under way to delineate the signaling pathway by which transcription of TGF-ß signaling is suppressed by AM and to determine whether such a signaling pathway governs both the maintenance of normal keratocytes and the inhibition of scar-forming myofibroblasts.
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Footnotes |
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Submitted for publication February 24, 2004; revised April 8, 2004; accepted May 12, 2004.
Disclosure: E.M. Espana, TissueTech, Inc. (F, E, P); T. Kawakita, TissueTech, Inc. (F, E); C.-Y. Liu, None; S.C.G. Tseng, TissueTech, Inc. (F, I, E, P)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Scheffer C. G. Tseng, Ocular Surface Center, 7000 SW 97 Avenue, Suite 213, Miami, FL 33173; stseng{at}ocularsurface.com.
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