Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs. The purpose of this study was to generate human iPSCs and direct them to limbal differentiation by maintaining them on natural substrata mimicking the native LESC niche, including feederless denuded human amniotic membrane (HAM) and de-epithelialized corneas. These iPSCs were generated with nonintegrating vectors from human primary limbal epithelial cells. This choice of parent cells was supposed to enhance limbal cell differentiation from iPSCs by partial retention of parental epigenetic signatures in iPSCs. When the gene methylation patterns were compared in iPSCs to parental LESCs using Illumina global methylation arrays, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs, suggesting retention of epigenetic memory during reprogramming. Limbal iPSCs cultured for 2 weeks on HAM developed markedly higher expression of putative LESC markers ABCG2, ΔNp63α, keratins 14, 15, and 17, N-cadherin, and TrkA than did fibroblast iPSCs. On HAM culture, the methylation profiles of select limbal iPSC genes (including NTRK1, coding for TrkA protein) became closer to the parental cells, but fibroblast iPSCs remained closer to parental fibroblasts. On denuded air-lifted corneas, limbal iPSCs even upregulated differentiated corneal keratins 3 and 12. These data emphasize the importance of the natural niche and limbal tissue of origin in generating iPSCs as a LESC source with translational potential for LSCD treatment.

PURPOSE:To determine the effi cacy of INTACS insertion using a femtosecond laser in the treatment of keratoconus and to compare it to the technique using a mechanical spreader.

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Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.

Purpose-To perform an immunohistochemical evaluation of corneas with INTACS for post-laser in situ keratomileusis (LASIK) keratectasia and keratoconus, obtained after corneal transplantation.Methods-Corneas from 1 patient with INTACS for post-LASIK keratectasia and 2 patients with INTACS for keratoconus were obtained within 3 hours after penetrating keratoplasty, and cryostat sections were analyzed by immunostaining for 35 extracellular matrix (ECM) components and proteinases.Results-In the stroma of all corneas next to an INTACS implant, ECM components typically associated with fibrosis were observed. These included tenascin-C, fibrillin-1, and types III, IV (α1/ α2 chains), and XIV collagen. Also, significant deposition of perlecan, nidogen-2, and cellular fibronectin was revealed in the same locations. The keratoconus cases displayed typical Bowman layer breaks and subepithelial fibrosis with deposition of various ECM components. In all cases, some keratocytes around INTACS were positive for specific proteinases associated with stromal remodeling, including cathepsins F and H, matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10. Staining for MMP-7 was variable; MMP-2 and MMP-9 were mostly negative. Patterns of type IV collagen α3, α4, and α6 chains; types VI and VIII collagen;α4, α5,β1, β2, and γ1 laminin chains; vitronectin; thrombospondin-1; urokinase; EMMPRIN; and cathepsins B and L were unchanged around INTACS in all 3 cases compared with normal. Recently, INTACS has acquired new interest as an alternative treatment of keratoconus. [5][6][7][8][9][10] The rationale is that INTACS not only flattens the central cornea but also corrects some of the astigmatic component. The corneal profile becomes more symmetrical, usually allowing better correction with glasses and contact lenses. Given that, for these patients, the alternative would be penetrating keratoplasty, INTACS may delay the need for this option. 10,11 Midstromal semicircular channels for INTACS ring insertion are created by a mechanical system with a blunt spiral device. 1,12,13 Despite the streamlined mechanistic procedure, attempts were made to improve control over the depth and shape of the channels. A femtosecond laser mounted on a delivery system capable of creating an intrastromal pattern of virtually any shape was recently used to create channels for INTACS insertion. 10 This method may be technically easier and more precise, reflecting better final outcomes. Conclusions-AbnormalKeratectasia is a rare but serious complication of laser vision correction of any refractive errors. It occurs mostly after LASIK but was also described after surface excimer laser ablation (photorefractive keratectomy). 14-16 It involves severe refractive changes mostly occurring several years after the surgery, which are caused by thinning and sagging of the corneal profile, similar to keratoconus.The etiology of this complication remains uncertain. In some cases, preexisting forme fruste keratoconus was documented. 17,18 Other cases could be caused by a me...