Corneal keratocytes: phenotypic and species differences in abundant protein expression and in vitro light-scattering

PURPOSE: Previous studies suggest that corneal haze after injury involves changes in the light-scattering properties of keratocytes that are possibly linked to the abundant expression of water-soluble proteins. The purpose of this study was to determine the protein expression pattern of keratocytes from different species and different cultured rabbit keratocyte phenotypes and to assess differences in light-scattering in vitro. METHODS: Water-soluble proteins were isolated from corneal epithelial cells and keratocytes of several species, including human (Hu), mouse (Mo), rabbit (Ra), chicken (Ch), and pig (P) and different cultured rabbit keratocyte phenotypes. Proteins were then characterized by SDS-PAGE, tryptic peptide sequence analysis, and Western blot analysis. Light-scattering and actin organization from cultured cells were determined with confocal reflectance and fluorescence microscopy, respectively. RESULTS: Protein expression patterns varied substantially between species and cell types, with five new abundantly expressed proteins identified including, LDH (Ra, Ch), G3PDH (Hu, Ch), pyruvate kinase (Ch), Annexin II (Ch), and protein disulfide isomerase (Ch). Different rabbit keratocyte phenotypes also showed different levels of expression of ALDH1A1 and TKT, with myofibroblasts showing the greatest reduction. Myofibroblasts showed significantly greater (P < 0.05) light-scattering but also showed the greatest organization of actin filaments. CONCLUSIONS: Abundant protein expression is a characteristic feature of corneal keratocytes that is lost when cells are phenotypically modulated in culture. Greater light-scattering by myofibroblasts also provides support for a link between cellular transparency and haze after injury that is possibly related to loss of protein expression or development of prominent actin filament bundles.     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

Myofibroblast differentiation of normal human keratocytes and hTERT,extended-life human corneal fibroblasts

PURPOSE: The purpose of this study was to determine whether TGFbeta induces myofibroblast differentiation in cultured human keratocytes and in telomerase (hTERT)-immortalized human corneal fibroblast cell lines. METHODS: Normal human corneal keratocytes were isolated from donor corneas of various ages and grown under serum-free (cultured keratocytes) or serum-added (corneal fibroblasts) conditions. Corneal fibroblasts were infected with the MPSV-hTERT retroviral vector, and selected clones were isolated and characterized by chromosomal karyotyping. The responses of normal cultured keratocytes and serum-starved corneal fibroblasts to TGFbeta in the presence or absence of Arg-Gly-Asp (RGD)-containing peptides and neutralizing antibodies to platelet-derived growth factor (PDGF) were characterized by immunocytochemistry, Western blot analysis, and real-time PCR, to identify assembly of actin filaments, formation of focal adhesions, and expression of alpha-smooth muscle actin (alpha-SMA). RESULTS: Treatment of cultured keratocytes with TGFbeta (1 ng/mL) induced cell spreading, assembly of actin filaments, formation of focal adhesions, and expression of alpha-SMA, which was blocked by the addition of RGD-containing peptides (100 microM). A similar response was identified in hTERT-expressing human corneal fibroblast cell lines, showing a 69-fold increase in alpha-SMA message. Furthermore, treatment of hTERT corneal fibroblasts with RGD or anti-PDGF inhibited myofibroblast differentiation. Karyotype analysis of hTERT corneal fibroblasts identified age-dependent chromosomal aberrations in cells of older donors but not in those of a 10-year-old donor. CONCLUSIONS: Induction of myofibroblast differentiation by TGFbeta in cultured human keratocytes and hTERT corneal fibroblasts occurs through a similar signal transduction pathway to that previously identified in the rabbit, which involves an autocrine PDGF feedback loop.     

Construction of a complete rabbit cornea substitute using a fibrin-agarose scaffold

PURPOSE: To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering. METHODS: Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy. RESULTS: All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium. CONCLUSIONS: These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.     

An investigation of removed cultivated epithelial transplants in patients after allocultivated corneal epithelial transplantation

OBJECTIVE: To investigate the ultrastructural changes of removed cultivated corneal epithelial transplants using scanning and transmission electron microscopy. METHODS: Allocultivated corneal epithelial transplantation, using an amniotic membrane carrier, was carried out on 3 patients. The primary diagnoses consisted of 1 with acute-phase chemical burn, one with drug-induced pseudopemphigoid, and 1 with Stevens-Johnson syndrome. After a period of several months the transplants were removed from these patients because of graft opacities. The removed transplants were then prepared for examination by scanning and transmission electron microscopy. RESULTS: In all 3 cases there was a similar pattern of findings: the amniotic membrane remained intact, although it had become partially vascularized and invaded by keratocytes. Inflammatory cells were present in the epithelial layer and within the amniotic membrane. Most of the amniotic membrane was covered by conjunctival epithelial cells and goblet cells. Only a few areas of normal cultivated corneal epithelial cells were found. CONCLUSIONS: We suggest that the process of allograft rejection is responsible for the corneal epithelial loss and that this is followed by conjunctival invasion onto the amniotic membrane.     

体外人羊膜培养兔角膜缘上皮细胞的细胞形态与连接复合体形成的研究

目的观察体外人羊膜上培养出的兔角膜缘上皮细胞形态特征及与羊膜基底膜连接复合体的形成。方法将15只新西兰白兔角膜缘组织（2mm×2mm）,于37℃ 5％ CO2孵箱中用1．2U／ml裂解酶Ⅱ处理20min,之后在人羊膜上皮面先行浸液培养2周,后行气-液培养2周。于培养期间,每周定期进行透射电镜检查,以观察连接复合体的形成过程,培养至第4周后,采用AE5免疫组织化学、高碘酸．希夫、苏木精-伊红等染色方法鉴定分化的上皮细胞,鉴别杯状细胞,观察细胞的形态特征。结果兔角膜缘上皮细胞体外培养10—14d可充满人羊膜面,对AE5免疫组织化学方法染色分化出的上皮细胞均呈阳性而高碘酸．希夫染色均呈阴性。苏木精-伊红染色显示从兔角膜缘上皮细胞分化出的上皮细胞与正常兔角膜上皮细胞相比无明显差异;自培养至第14天均未见与羊膜基底膜的连接复合体形成;于第21天时仅观察到初始阶段的连接复合体,第28天时仍未见明显变化。结论于体外人羊膜上培养的兔角膜缘上皮细胞分化出的细胞是角膜上皮细胞,其形态与正常兔角膜上皮细胞相同,并与羊膜基底膜仅形成初始阶段的连接复合体。     

Differential regulation of fibronectin synthesis in three different types of corneal cells

The production of fibronectin (FN) and its response to serum or epidermal growth factor (EGF) were investigated in three different types of rabbit corneal cells cultured in vitro. The corneal epithelial cells, stromal fibroblasts (keratocytes) and endothelial cells were separately cultured in different media: basic medium containing minimal serum (0.5%), basic medium with supplementary serum at a final concentration of 10% and basic medium with 100 ng/ml EGF, respectively. FN production by each type of cell was examined either by the immunofluorescent staining method or by the metabolic labeling method followed by immunoprecipitation of FN in the culture medium. Each type of corneal cell produced and secreted FN. FN secretion into the culture medium by keratocytes and by endothelial cells was enhanced by the addition of EGF. However, FN secretion by epithelial cells was lowered by the additional serum or EGF. Furthermore, when the epithelial cells were cultured in the basic medium, DNA synthesis was low but FN secretion was high. These results suggest that the control mechanism of FN production differs between epithelial cells and keratocytes or endothelial cells.     

TGFbeta induced myofibroblast differentiation of rabbit keratocytes requires synergistic TGFbeta,PDGF and integrin signaling

There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFbeta). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFbeta. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFbeta1 stimulated cell cycle entry of serum-free cultured rabbit corneal keratocytes. AntiPDGF treatment also markedly reduced the TGFbeta1-induced intracellular actin filament re-organization, fibronectin fibril assembly, and focal contact formation as well as reducing by 80% the expression of alpha-smooth muscle (alpha-SM) specific isoform of actin characteristic of myofibroblast differentiation. Although PDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFbeta1 as did quiescent keratocytes. Furthermore, blocking TGFbeta1 induction of myofibroblast differentiation with the Arg-Gly-Asp containing peptide, GRGDdSP, for 3 days followed by allowing progression of myofibroblast differentiation by removing GRGDdSP did not change the temporal response or tyrosine phosphorylation cascade (2-72 hr) leading to myofibroblast differentiation. Nor did PDGF treatment of keratocytes reverse the RGD blockade of TGFbeta1 induced myofibroblast differentiation. Overall these cumulative findings indicate that myofibroblast differentiation in the rabbit corneal keratocyte requires synergistic growth factor/integrin signaling involving TGFbeta, PDGF, and the fibronectin receptor. Additionally, the similar TGFbeta1 temporal response of PDGF-stimulated compared to nai;ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.     

Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes

Transparency is essential for normal corneal function. Recent studies suggest that corneal cells express high levels of so-called corneal crystallins, such as aldehyde dehydrogenase (ALDH) and transketolase (TKT) that contribute to maintaining cellular transparency. Stromal injury leads to the appearance of repair phenotype keratocytes, the corneal fibroblast and myofibroblast. Previous studies on keratocytes from species such as bovine and rabbit indicate that the transformation from the normal to repair phenotype is accompanied by a loss of corneal crystallin expression, which may be associated with loss of cellular transparency. Here we investigated if a similar loss occurs with human keratocyte repair phenotypes. Human corneal epithelial cells were collected by scraping and keratocytes were isolated by collagenase digestion from cadaveric corneas. The cells were either processed immediately (freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. RT-PCR, western blotting and immunolabeling were used to detect mRNA and protein expression of ALDH isozymes and TKT. ALDH enzyme activity was also quantitated and immunolabeling was performed to determine the expression of ALDH3A1 in human corneal tissue sections from normal and diseased corneas. Human corneal keratocytes isolated from three donors expressed ALDH1A1 and ALDH3A1 mRNA, and one donor also expressed ALDH2 and TKT. Corneal epithelial cells expressed ALDH1A1, ALDH2, ALDH3A1 and TKT. Compared to normal keratocytes, corneal fibroblast expression of ALDH3A1 mRNA was reduced by 27% (n=5). ALDH3A1 protein expression as detected by western blotting was markedly reduced in passage zero fibroblasts and undetectable in higher passages (n=3). TKT protein expression was reduced in fibroblasts compared to keratocytes (n=2). ALDH3A1 enzyme activity was not detectable in corneal fibroblasts (n=6) but was readily detected in corneal epithelial cells (0.29+/-0.1U/mg protein, n=4) and keratocytes (0.05+/-0.009U/mg protein, n=7). ALDH3A1 expression was also reduced in corneal fibroblasts and myofibroblasts as determined by immunolabeling of the cells in culture (n=3) and in diseased corneal tissues in situ (n=2). We conclude that expression of the crystallin ALDH3A1 is decreased in repair phenotype human keratocytes, compared to normal human keratocytes. Extrapolating from studies of bovine and rabbit, the reduced expression of ALDH3A1 may contribute to the loss of corneal transparency experienced by human patients after injury and refractive surgeries.     

Actin filament localization and distribution in the young adult mouse cornea: a correlative immunofluorescent and cytochemical study

The localization and distribution of actin filaments was determined in the corneal epithelium and endothelium of the young adult Swiss Webster mouse by correlative indirect immunofluorescence using rabbit anti-skeletal muscle actin antiserum and by in situ labelling with heavy meromyosin subfragment-1 (HMM-S1). A diffuse fluorescent staining was observed in the cytoplasm of epithelial and endothelial cells and in stromal keratocytes. In addition, a highly fluorescent punctate cytoplasmic staining was seen only in the corneal epithelium. Ultrastructurally, HMM-S1 decorated actin filament bundles were generally distributed within the cytoplasm of glycerinated superficial, wing and basal epithelial cells and within pedicle-like processes on the posterior surface of superficial and wing cells. Actin filament bundles also were seen in close association with intermediate filament bundles in the epithelium. HMM-S1 labelled filaments were observed in the endothelial cytoplasm anteriorly near Descemet's membrane, posteriorly near the anterior chamber and adjacent to lateral cell borders. Actin filaments in epithelial and endothelial cells appeared to insert into the plasma membrane.     

Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits

PURPOSE: To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. METHODS: An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. RESULTS: A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. CONCLUSION: Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.     

Localization of fibronectin and actin in cultured rabbit keratocytes

Migration of activated keratocytes toward the corneal stromal wound is one of the most important processes of successful healing. To understand the motility of keratocytes and the interaction of fibronectin and intracellular actin filaments, we cultured rabbit corneal keratocytes and studied dynamic movements of the cells by time-lapse cinematography. We also examined the changes in the localization of fibronectin and actin using double staining immunofluorescent microscopy. The cultured keratocytes first attached to the substratum in round globular shape and then spread with many extending processes. In the early stage of the cultivation, fibronectin was observed inside the cells. Later, fibronectin was observed outside the cells, suggesting formation of the extracellular matrix. When keratocytes spread, actin was observed as a stress fiber inside the cells. At the edge of the cellular processes, a close interaction between fibronectin and actin was observed. The present results demonstrated that cultured keratocytes had active motility and that there were close interactions between the extracellular fibronectin and intracellular actin filaments. The organization of fibrillar actin filaments (F-actin) might be affected by the binding of extracellular fibronectin to the cell surface receptor for fibronectin.     

培养角膜缘干细胞羊膜移植治疗碱烧伤动物的实验研究

目的 观察培养生长于羊膜的角膜缘干细胞移植的治疗角膜缘碱烧伤伤的效果。方法 将兔角膜缘干细胞在的代培养后接种于羊膜,对新西兰大白兔角膜缘碱烧伤动物模型行角膜缘干细胞羊膜移植术,并对治疗后的角膜进行临床及病理学检查。结果 体外培养的兔角膜缘士细胞可在羊膜上继续增殖、分化为密集的角膜上皮细胞层;角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮完整基质细胞浸润减轻、新生血管减少。组织病理学染色证实,角膜缘     

Comparison of cell-suspension and explant culture of rabbit limbal epithelial cells

Currently, most investigators directly use limbal explants to culture corneal epithelial cells. However, it has not been identified that limbal stem cells do readily migrate from the limbal explants onto culture plate or amniotic membrane carrier. In this study a cell-suspension culture system for rabbit limbal stem cells was developed and compared with the direct explant method in the aspect of stem cells content in the culture system. Rabbit limbal epithelial cells were dissociated from rabbit eyes by dispase and single cell suspension was made for cell-suspension culture. DeltaNp63 expression of cultured rabbit limbal epithelial cells by cell-suspension technique and explant technique was detected. In cell-suspension culture, isolated cell-suspension was evaluated by flow cytometric analysis for vimentin expression and residual limbal tissue after dispase treatment was examined by scanning electron microscopy. In limbal epithelial cells suspension less than 5% cells were vimentin positive. Examination of residual limbal tissue confirmed that all the limbal epithelial cells had been removed. Histological examination revealed that with cell-suspension culture the cultured epithelial cells could differentiate better than with explant technique. In cells cultured with cell-suspension, there were much more cells expressing DeltaNp63 than in explant cultured cells. In cells cultured with explants, most of DeltaNp63 labelling cells mustered around the explants, and peripheral cells on the slides were DeltaNp63 negative. These results suggested that with pure limbal epithelial cells suspension including basal cells, which could directly enter into culture system, cell-suspension culture technique was significantly superior to explant culture technique in terms of stem cells content.     

Involvement of S100A4 in stromal fibroblasts of the regenerating cornea

PURPOSE. S100A4 is a member of the S100 family of calcium-binding proteins. Members of the S100 family have been implicated in a variety of cellular events, including growth, signaling, differentiation, and motility. It has been suggested that S100A4 modulates cell shape and motility by interacting with components of the cytoskeleton. In the present study, the distribution patterns of S100A4 were investigated in normal and regenerating mouse corneas. METHODS. Rabbit cDNA libraries were prepared from cultures of corneal fibroblasts. S100A4 was identified as the most abundant message present. Expression of S100A4 in the cornea was determined using Northern blot analysis, in situ hybridization, and immunohistochemistry. Distribution patterns of S100A4 in primary corneal fibroblast cultures treated with either FGF-2/heparin or TGFbeta1 were analyzed by immunofluorescence. RESULTS. S100A4 mRNA was rarely detected in keratocytes or epithelial cells of the normal rabbit cornea. Likewise, S100A4 antigen was not found in normal mouse corneas. However, after removal of the corneal epithelium, fibroblasts are activated and had readily detectable S100A4 expression 6 days after wounding. In the in vitro equivalent of activated keratocytes, cultured rabbit corneal fibroblasts, S100A4 was restricted to the cytoplasm. In contrast, in cultures treated with TGFbeta1, which induces a myofibroblast phenotype, more than 90% of the cells showed a nuclear localization of S100A4. CONCLUSIONS. The findings show that S100A4 is expressed in the keratocyte phenotypes that appear in stromal tissue of corneas recovering from damage, the fibroblasts, and myofibroblasts. Its expression and distinct subcellular redistribution patterns suggest that S100A4 may be involved in the interconversions that occur between keratocytes, fibroblasts, and myofibroblasts during corneal wound healing.     

In vivo laser confocal microscopic analysis of human corneal epithelial sheet cultured on amniotic membrane.

BACKGROUND AND OBJECTIVE: Corneal epithelial sheet cultured on amniotic membrane ex vivo has been developed for the treatment of severe ocular surface disorders. In this study, human corneal epithelial sheets cultured on amniotic membranes were analyzed using a laser scanning confocal microscope. MATERIALS AND METHODS: Human corneal epithelial cells were cultured on four sheets of amniotic membrane, and they were examined layer by layer using the laser scanning confocal microscope. RESULTS: The superficial layer cells were mosaic in appearance. In the superficial layer, focal areas of slender and elongated cells were noted. In addition, highly reflective materials were noted. Basal epithelial cells of the corneal epithelium sheet at a slightly deeper plane displayed mosaic pattern with smaller cells. At a slightly deeper plane, the basement membrane of amnion showed relatively bright homogeneity. CONCLUSION: The laser scanning confocal microscope can examine the morphology of human corneal epithelial cells cultured on amniotic membranes.     

兔角膜基质细胞在壳聚糖胶原共混膜体外培养研究

目的　探讨壳聚糖 胶原共混膜作为载体体外培养兔角膜基质细胞的可行性。方法　先同时消化角膜上皮及内皮层 ,然后刮去上皮、内皮、前弹力层和后弹力层 ,将剩余的基质层剪碎消化 ,接种在壳聚糖 胶原共混膜上 ,通过间接免疫荧光细胞化学染色对培养的细胞进行鉴别。结果　角膜基质细胞应用消化培养法 4h后部分基质细胞与壳聚糖 胶原共混膜有贴壁现象出现 ,细胞呈梭形。培养 2 4h后 ,基质细胞呈纺锤形且透明 ,此后细胞分裂增殖越来越多并向周围延伸 ,培养第 6天细胞已经达到完全融合状态 ,呈梭形 ,排列比较整齐。在 6d左右达到 10 0 %融合状态 ,间接免疫荧光细胞化学染色、Vim染色、胞浆染色阳性。结论　传代的细胞具有角膜基质细胞的生物特性 ,壳聚糖 胶原共混膜适合角膜基质细胞传代培养     

Injured corneal epithelial cells promote myodifferentiation of corneal fibroblasts

PURPOSE: To determine whether injured corneal epithelial cells stimulate myodifferentiation in corneal fibroblasts and whether transforming growth factor (TGF)-beta is involved. METHODS: Rabbit corneal fibroblasts were cultured on collagen gel, with or without cocultured corneal epithelial cells or with partially scraped epithelial cells, on a companion plate separated by a permeable membrane. To evaluate fibroblast-induced gel contraction, gel thickness was measured daily relative to the original thickness. Total fibroblasts on the gel were counted. Myofibroblasts were counted by using immunocytochemical identification with anti-alpha-smooth muscle actin (alpha-SMA). TGF-beta was assayed in the media on days 3 and 6. These procedures also were performed in the presence of anti-TGF-beta antibody. RESULTS: Gel contraction, alpha-SMA-positive cells, and total cell number were significantly greater on gels with injured epithelial cells than on gels without epithelial cells or with uninjured epithelial cells, as was TGF-beta concentration in the media. Anti-TGF-beta antibody eliminated these differences. CONCLUSIONS: Injured epithelial cells stimulate myodifferentiation in fibroblasts through one or more soluble factors, including TGF-beta.