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Preservation and Expansion of the Primate Keratocyte Phenotype by Downregulating TGF-␤ Signaling in a Low-Calcium, Serum-Free Medium

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Preservation and Expansion of the Primate Keratocyte Phenotype by Downregulating TGF-␤ Signaling in a Low-Calcium, Serum-Free Medium Preservation and Expansion of the Primate Keratocyte Phenotype by Downregulating TGF-␤ Signaling in a Low-Calcium, Serum-Free Medium Tetsuya Kawakita,1 Edgar M. Espana,1 Hua He,1 Robert Smiddy,1 Jean-Marie Parel,2 Chia-Yang Liu,2 and Scheffer C. G. Tseng1 PURPOSE. To demonstrate whether the original keratocyte phe- regulating Smad-mediated TGF-␤ signaling. (Invest Ophthalmol notype is maintained with proliferative activity by suppressing Vis Sci. 2006;47:1918–1927) DOI:10.1167/iovs.05-1040 TGF-␤ signaling in rhesus monkey keratocytes expanded in a serum-free and low-[Ca2ϩ] medium. METHODS. Rhesus monkey keratocytes were isolated from cen- eratocytes, a unique population of neural crest-derived tral corneal buttons by collagenase digestion for 16 hours, Kcells embedded in the corneal stroma, play a major role in seeded on plastic in Dulbecco’s modified Eagle’s medium maintaining corneal transparency. They are mitotically quies- (DMEM) containing insulin-transferrin-sodium selenite (ITS) cent, exhibit a dendritic morphology with extensive intercel- supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/ lular contacts,1,2 and express keratan sulfate-containing proteo- 10% FBS), or in a defined keratinocyte serum-free medium glycans (KSPGs),3,4 aldehyde dehydrogenase (ALDH)4 and (KSFM). After confluence, cells in KSFM were continuously CD34.5–7 When a scar forms during corneal wound healing, subcultured at a 1-to-3 split. Cellular proliferation was analyzed keratocytes turn into fibroblasts by losing the aforementioned by immunostaining for Ki67 and the MTT assay. The cellular morphology and downregulating the expression of KSPG8,9 phenotype was determined by immunostaining for aldehyde and CD34,5 and eventually they differentiate into myofibro- dehydrogenase (ALDH), keratocan, and CD34 and by the ex- blasts10,11 that express ␣-smooth muscle actin (␣-SMA), fi- pression of keratocan promoter-driven enhanced cyan fluores- bronectin, and biglycan.4 cent protein (ECFP). The stability of the keratocyte phenotype The aforementioned abnormal phenotypic changes can be was examined by switching KSFM to DMEM/ITS and DMEM/ observed in vitro on plastic dishes by adding fetal bovine serum 10% FBS. TGF-␤ signaling was monitored by measuring the (FBS). For example, the characteristic dendritic morphology of promoter activity of TGF-␤1, -␤2, and -␤ RII after transient bovine,12 rabbit,13,14 and human7,15 keratocytes is maintained adenoviral transfection, and cytolocalization of Smad2 and Smad4. in a serum-free medium. Nevertheless, when FBS is added, cells take on a flattened fibroblastic morphology and lose the ex- RESULTS. In KSFM, monkey keratocytes proliferated while main- pression of KSPG, including corneal stroma-specific kerato- taining the expression of keratocan, CD34, and ALDH proteins can.4,7,15–19 Unfortunately, keratocytes cultured in the serum- and keratocan promoter-driven ECFP for at least 15 passages. free medium do not proliferate. The nuclear accumulation of Smad2 and Smad4 and the pro- Therefore, expanding keratocytes while maintaining their moter activities of TGF-␤1 and -␤ RII were significantly down- normal phenotype in vitro is very desirable. Because FBS con- regulated in KSFM compared with DMEM/10% FBS. In KSFM, ␤ 20 an increase of [Ca2ϩ] to 1.8 mM and addition of 10% FBS tains TGF- , which promotes myofibroblast differentia- 4,14,21,22 ␤ synergistically downregulated the keratocan promoter activity, tion, many have speculated that TGF- signaling is facilitated Smad2 and Smad4 nuclear translocation, and upregu- responsible for promoting myofibroblast differentiation from lated TGF-␤1 and -␤ RII promoter activities. keratocytes. It remains unknown whether suppression of TGF-␤ signaling is not only sufficient to prevent myofibroblast CONCLUSIONS. The normal monkey keratocyte phenotype can differentiation but also essential for maintaining the keratocyte be maintained in a low-calcium, serum-free medium by down- phenotype. Because expression of the TGF-␤2, -␤3, and -␤ RII transcripts is suppressed when human corneal and limbal fi- 23 1 broblasts and human conjunctival and pterygium fibro- From TissueTech, Inc., Ocular Surface Center, Miami, Florida; 24 and the 2Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, blasts were cultured on human amniotic membrane (AM) University of Miami School of Medicine, Miami, Florida. stroma, we used AM as a culturing substrate to maintain a Supported by TissueTech, Inc., Ocular Surface Research and Ed- dendritic morphology and expression of keratocan and CD34 ucation Foundation, Miami, Florida; National Eye Institute Grants by human keratocytes while continuously stimulating them to EY06819 and EY15735 (SCGT) and EY12486 (C-YL); the Florida Lions proliferate in a medium containing 10% FBS.7,15 Extracellular Eye Bank (J-MP); and the Henri and Flore Lesieur Foundation (J-MP). calcium concentration ([Ca2ϩ]) significantly affects TGF-␤ sig- Submitted for publication August 7, 2005; revised December 6, 2ϩ 2005, and January 10, 2006; accepted March 23, 2006. naling. For example, increased [Ca ] promotes expression of ␤ 25 Disclosure: T. Kawakita, TissueTech, Inc. (E, F); E.M. Espana, TGF- in human vascular endothelial cells. Chelation of 2ϩ 2ϩ TissueTech, Inc. (E, F); H. He, TissueTech, Inc. (E, F); R. Smiddy, extracellular [Ca ] blocks TGF-␤-mediated [Ca ] influx TissueTech, Inc. (E, F); J.-M. Parel, TissueTech, Inc. (F); C.-Y. Liu, and calcineurin (calcium-dependent signaling intermediate) TissueTech, Inc. (F); S.C.G. Tseng, TissueTech, Inc. (E, F) activity,26 and inhibits TGF-␤-mediated ␣-SMA promoter signal- The publication costs of this article were defrayed in part by page ing in human embryonic lung fibroblasts.27 With this [Ca2ϩ] charge payment. This article must therefore be marked “advertise- effect in mind, we examined in the current study whether the ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Scheffer C. G. Tseng, Ocular Sur- phenotype of keratocytes can also be maintained on plastic by 2ϩ face Center, 7000 SW 97 Avenue, Suite 213, Miami, FL 33176; suppressing TGF-␤ signaling with a low-[Ca ], serum-free [email protected]. medium supplemented with growth factors. Investigative Ophthalmology & Visual Science, May 2006, Vol. 47, No. 5 1918 Copyright © Association for Research in Vision and Ophthalmology Downloaded from jov.arvojournals.org on 09/25/2021 IOVS, May 2006, Vol. 47, No. 5 Preservation of Primate Keratocyte Phenotype 1919 MATERIALS AND METHODS plement (DMEM/ITS; cat. no. 41400-045; Invitrogen-Gibco) or 10% FBS (DMEM/10% FBS). Reagents When the primary culture on plastic reached 80% confluence, cells were rendered into single cells by incubation in BSS containing 0.25% The tissue culture plastic plates (96-well and 6-well) were purchased trypsin/1 mM EDTA at 37°C for 1 to 5 minutes, and the enzymatic from BD Biosciences (Lincoln Park, NJ); collagenase A and dispase II reaction was stopped by adding soybean-trypsin inhibitor. After they powder from Roche (Indianapolis, IN); amphotericin B, Dulbecco’s were centrifuged at 800g for 5 minutes, the cells were resuspended in modified Eagle’s medium (DMEM), defined keratinocyte serum-free- KSFM, subdivided into three equal parts, and seeded on plastic dishes. medium (KSFM), FBS, gentamicin, Hanks ’ balanced salt solution They were cultured in KSFM continuously until use. Keratocytes were (HBSS), HEPES buffer, phosphate-buffered saline (PBS), soybean tryp- similarly isolated from mouse, rabbit, and human corneas and cultured sin inhibitor, and 0.25% trypsin/1 mM EDTA were purchased from in KSFM for comparison. Invitrogen-Gibco (Grand Island, NY); A cell-viability–cytotoxicity kit ␤ (Live/Dead) from Invitrogen (Eugene, OR); endo- -galactosidase was Cell Proliferation from Seikagaku (Tokyo, Japan); optimal cutting temperature (OCT) from Sakura Finetek (Torrance, CA); and a cell-proliferation assay Cell proliferation in KSFM was verified by subculturing primary cells in (MTT) from Roche. Other reagents and chemicals including bovine DMEM/ITS, DMEM/10% FBS, or KSFM at a density of 3000 cells per serum albumin (BSA) and insulin-transferrin-sodium selenite (ITS) me- 96-well plastic dish, and subjected at days 3 and 7 to the MTT assay dium supplement were from Sigma-Aldrich. The monoclonal antibod- (Promega Corp., Madison, WI), according to the manufacturer’s in- ies against CD34 (QBEnd 10) and Ki67 (MIB-1) were from Dako structions. Briefly, this assay measures mitochondrial dehydrogenase (Carpinteria, CA), and that against ALDH (clone 44) was from BD enzyme activity using the substrate of 3-(4,5-dimethylthiazol-2-yl)-2,5- Biosciences (San Jose, CA). The polyclonal antibodies against Smad2 diphenyltetrazolium bromide at absorbance of 50 nm, to reflect the (S-20) and Smad4 (C-20) were from Santa Cruz Biotechnology (Santa proliferative activity of viable cells. Using the culture medium alone as Cruz, CA). the negative control, we had validated this assay by establishing a linear correlation between 2,500 and 10,000 passage-2 murine corneal fibro- blasts (data not shown). Cells at day 7 were also immunostained with Preparation of an Epitope-Specific, Polyclonal, an anti-Ki67 antibody (1:100). The number of Ki67-positive nuclei was Anti-human Keratocan Antibody randomly measured in 10 fields under high magnification (400ϫ) for Rabbit anti-human keratocan (hKera) peptide antiserum was obtained each culture and the ratio of positive cells to total cells calculated in from EvoQuest Custom Antibody Services
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