Julie T. Daniels

Characterization of the Limbal Epithelial Stem Cell Niche: Novel Imaging Techniques Permit In Vivo Observation and Targeted Biopsy of Limbal Epithelial Stem Cells

It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State‐of‐the‐art imaging techniques were used to construct a three‐dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63α and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies.

Corneal stem cells in review

The cornea provides the eye with protection and the refractive properties essential for visual acuity. The transparent epithelium is highly specialized with basal and stratified squamous cells that are renewed throughout life from a stem cell population. The stem cells are thought to reside at the corneal limbus and may be maintained by a variety of intrinsic and extrinsic factors such as the local environment, survival factors, and cytokines. A number of markers have been localized to the limbus in an attempt to identify stem cells; however, definite stem cell identification remains elusive. During homeostasis and following injury to the corneal epithelium, the limbal stem cells divide to produce daughter transient amplifying cells that proliferate, migrate, and differentiate to replace lost cells. However, this cannot occur if the stem cell population is depleted. Limbal stem cell deficiency then results in corneal re‐epithelialization by the neighboring conjunctiva, causing pain, poor vision, and even blindness. This review will focus on corneal epithelial stem cells in ocular surface repair and regeneration. The current knowledge of stem cell biology in the corneal epithelium, clinical consequences of stem cell deficiency, and therapeutic strategies aimed at reversing stem cell deficiency will be discussed.

Ex Vivo Expansion and Transplantation of Limbal Epithelial Stem Cells

OBJECTIVE To determine, using objective measures, the outcome of ex vivo cultured limbal epithelial stem cell (LESC) transplantation performed in compliance with good manufacturing practice using a novel culture system without 3T3 feeder cells. DESIGN Prospective, noncomparative, interventional case series. PARTICIPANTS Ten eyes of 10 patients with profound LESC deficiency arising from chemical injury (4 eyes), aniridia (3 eyes), ectodermal dysplasia (1 eye), Reigers anomaly with Pax6 haploinsufficiency (1 eye), and unknown cause (1 eye). METHODS Allogeneic (7 eyes) or autologous (3 eyes) corneal LESCs were cultured on human amniotic membrane. Tissue was transplanted to the recipient eye after superficial keratectomy. Impression cytology and confocal microscopy were performed 6 months after surgery with clinical follow-up to 13 months. Success was defined as an improvement in the defined clinical parameters of LESC deficiency, an improvement in visual acuity, the restoration of a more normal corneal phenotype on impression cytology, and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. MAIN OUTCOME MEASURES Clinical parameters of LESC deficiency (loss of epithelial transparency, superficial corneal vascularization, epithelial irregularity, and epithelial breakdown), visual acuity, impression cytology and cytokeratin expression profiles, and in vivo confocal corneal confocal microscopy. RESULTS The success rate using this technique was 60% (autografts 33%, allografts 71%). All patients with a successful outcome experienced an improvement in visual acuity of >/=2 lines Snellen acuity. Preoperatively, CK3+ and CK19+ cells accounted for 12+/-2.4% (mean +/- standard error of the mean) and 80+/-2.15% of cells, respectively, whereas postoperatively these accounted for 69+/-6.43% (P<0.0001) and 30+/-6.34% (P<0.0001) of cells, respectively. Goblet cells accounted for 8+/-1.19% of cells preoperatively and 1+/-0.35% of cells postoperatively (P<0.0001). CONCLUSIONS These data demonstrate that it is possible to culture LESCs ex vivo in compliance with good manufacturing practice regulations. A set of objective outcome measures that confirm the efficiency of this technique in treating LESC deficiency is described. The widespread use of such standardized and objective outcome measures would facilitate a comparison between the different culture methods in use.

Matrix Metalloproteinases in Disease and Repair Processes in the Anterior Segment

The pathogenesis of many anterior segment disorders and ocular complications following surgery are secondary to the wound healing response. The extent of clinical damage observed is closely related to the amount of scarring and tissue contraction. Matrix metalloproteinases (MMPs) are a family of enzymes that play a vital role in all stages of the wound healing process. They degrade all extracellular matrix components and also have the ability to synthesize collagen and extracellular matrix members, and are therefore important in the remodeling of a wound. Overexpression of MMPs results in excessive extracellular matrix degradation, leading to tissue destruction and loss of organ function. In the case of the anterior segment, this may mean the loss of visual function. This review focuses on the role MMPs have in the development of various anterior segment disorders. The importance of MMPs in the wound healing response and its potential modulation to manipulate the scarring response is being recognized, and current developments will be described.

Skin and oral fibroblasts exhibit phenotypic differences in extracellular matrix reorganization and matrix metalloproteinase activity

Background Oral mucosal wounds are characterized by rapid re‐epithelialization and remodelling. In vitro, oral mucosal fibroblasts exhibit a fetal phenotype with increased extracellular matrix reorganizational ability, migration and experimental wound repopulation when compared with skin fibroblasts.

Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture

We describe, for the first time, the use of cellular plastic compressed collagen as a substrate for human limbal epithelial cell expansion and stratification. The characteristics of expanded limbal epithelial cells on either acellular collagen constructs or those containing human limbal fibroblasts were compared to a human central cornea control. After compression, human fibroblasts in collagen constructs remained viable and limbal epithelial cells were successfully expanded on the surface. After airlifting, a multilayered epithelium formed with epithelial cell morphology very similar to that of cells in the central cornea. Immunochemical staining revealed expression of basement membrane proteins and differentiated epithelial cell markers found in native central cornea. Ultrastructural analysis revealed cells on collagen constructs had many features similar to central cornea, including polygonal, tightly opposed surface epithelial cells with microvilli and numerous desmosomes at cell-cell junctions. Taken together, these data demonstrate that plastic compressed collagen constructs can form the basis of a biomimetic tissue model for in vitro testing and could potentially provide a suitable alternative to amniotic membrane as a substrate for limbal epithelial cell transplantation.

Modulation of wound healing after glaucoma surgery.

The healing process after glaucoma filtration is the main determinant of surgical failure and, even more important, the final intraocular pressure. The ability to fully control wound healing may ultimately give us the ability to set the intraocular pressure in the low teens for all patients undergoing glaucoma filtration surgery. The authors review the changes in how to use antimetabolites to improve safety, and many of the exciting new areas of progress, including growth factor neutralization and future molecular therapies to control wound healing.

Mediation of Transforming Growth Factor-β1-Stimulated Matrix Contraction by Fibroblasts: A Role for Connective Tissue Growth Factor in Contractile Scarring

Excessive cell-mediated tissue contraction after injury can lead to morbid contractile scarring in the body. In the eye this can cause blindness because of posterior capsule opacification, proliferative vitroretinopathy, failure of glaucoma filtration surgery, and corneal haze. During repair, transforming growth factor (TGF)-beta and connective tissue growth factor (CTGF) genes are co-ordinately expressed. Although TGF-beta and CTGF stimulate new matrix deposition, their role and regulation during contractile scarring is unknown. In this study, an in vitro model of collagen matrix contraction culminating from tractional forces generated by fibroblasts showed that both TGF-beta(1) and CTGF-stimulated contraction. Using a specific anti-sense oligodeoxynucleotide to CTGF the procontractile activity of TGF-beta(1) was found to be mediated by CTGF. During contraction fibroblasts produced similar levels of matrix metalloproteinases (MMPs)-2 and -9 with TGF-beta(1) or CTGF and a modest increase in MMP-1 with CTGF only (indicated by zymography and enzyme-linked immunosorbent assay). The requirement of MMPs for contraction was demonstrated using a broad-spectrum synthetic inhibitor. This study demonstrates a new function for CTGF in mediating matrix contraction by fibroblasts involving MMPs and suggests a novel regulatory mechanism for TGF-beta-stimulated contraction. Inhibition of CTGF activity or gene transcription could be a suitable target for anti-scarring therapies.

The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells.

Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (< or =4% in both intact and acellular groups) and ABCG2 (< or =3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (< or =6%) and ABCG2 (< or =8%). HAM frozen at -80 degrees C in Hanks Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (< or =68%) and ABCG2 (< or =62%) expression (p<0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.

In sickness and in health: Corneal epithelial stem cell biology, pathology and therapy

Our window to the world is provided by the cornea on the front surface of the eye. The integrity and functionality of the outermost corneal epithelium is essential for vision. A population of limbal epithelial stem cells (LESCs) are responsible for maintaining the epithelium throughout life by providing a constant supply of daughter cells that replenish those constantly lost from the ocular surface during normal wear and tear and following injury. LESC deficiency leads to corneal opacification, inflammation, vascularization and discomfort (Daniels et al., 2001, 2007). Cultured LESC delivery is one of several examples of successful adult stem cell therapy in patients. The clinical precedence for use of stem cell therapy and the accessibility of the transparent stem cell niche make the cornea a unique model for the study of adult stem cells in physiological conditions as well as in disease.