[Show abstract][Hide abstract] ABSTRACT: Limbal stem cell deficiency (LSCD) is an eye disorder in which the stem cells responsible for forming the surface skin of the cornea are destroyed by disease. This results in pain, loss of vision, and a cosmetically unpleasant appearance. Many new treatments, including stem cell therapies, are emerging for the treatment of this condition, but assessment of these new technologies is severely hampered by the lack of biomarkers for this disease or validated tools for assessing its severity. The aims of this study were to design and test the reliability of a tool for grading LSCD, to define a set of core outcome measures for use in evaluating treatments for this condition, and to demonstrate their utility. This was achieved by using our defined outcome set (which included the Clinical Outcome Assessment in Surgical Trials of Limbal stem cell deficiency [COASTL] tool) to evaluate the 3-year outcomes for allogeneic ex vivo cultivated limbal epithelial transplantation (allo-CLET) in patients who had bilateral total LSCD secondary to aniridia or Stevens-Johnson syndrome. The results demonstrate that our new grading tool for LSCD, the COASTL tool, is reliable and repeatable, and that improvements in the biomarkers used in this tool correlate positively with improvements in visual acuity. The COASTL tool showed that following allo-CLET there was a decrease in LSCD severity and an increase in visual acuity up to 12 months post-treatment, but thereafter LSCD severity and visual acuity progressively deteriorated.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To investigate signal transduction pathways for connective tissue growth factor (CTGF) in human corneal fibroblasts (HCF).
Expression of 75 kinases in cultures of serum-starved (HCF) were investigated using protein kinase screens, and changes in levels of phosphorylation of 31 different phosphoproteins were determined at 0, 5, and 15 minutes after treatment with CTGF. Levels of phosphorylation of three signal transducing phosphoproteins (extracellular regulated kinase 1 [ERK1], extracellular regulated kinase 2 [ERK2] [MAPKs], and signal transducer and activator of transcription 3 [STAT3]) were measured at nine time points after exposure to CTGF using Western immunoblots. Inhibition of Ras, MEK1/2 (MAPKK), and ERK1/2, on CTGF-stimulated fibroblast proliferation and collagen gel contraction was assessed using selective inhibitors farnesylthiosalicylic acid, PD-98059, and SB203580, respectively.
Thirty two of the 75 kinases (43%) evaluated by the kinase screen were detected in extracts of quiescent HCF, suggesting these kinases are available to respond acutely to CTGF exposure. Addition of CTGF increased levels of phosphorylation of five phosphoproteins (ERK1 and 2, MEK1/2 [MAPKK], STAT3, and SAPK/JNK), and decreased levels of phosphorylation of 14 phosphoproteins (including protein kinases B and C) after 5 and 15 minutes. Further analysis of ERK1 and 2 and STAT3 phosphorylation showed rapid increases within 1 minute of CTGF exposure that peaked between 5 and 10 minutes then returned to pretreatment levels by 30 minutes. Treatment of HCF with selective inhibitors of Ras, MEK 1/2, and ERK1/2 individually blocked both CTGF induced cell proliferation, and collagen gel contraction.
Results from protein kinase screens and selective kinase inhibitors demonstrate Ras/MEK/ERK/STAT3 pathway is required for CTGF signaling in HCF.
[Show abstract][Hide abstract] ABSTRACT: 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 (≤4% in both intact and acellular groups) and ABCG2 (≤3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (≤6%) and ABCG2 (≤8%). HAM frozen at −80 °C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (≤68%) and ABCG2 (≤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.
[Show abstract][Hide abstract] ABSTRACT: The cornea on the front surface of the eye is our window to the world, hence maintenance of corneal tissue transparency is essential for vision. The integrity and functionality of the outermost corneal layer, the epithelium, plays a key role in refraction of light on to the retina at the back of the eye. Like other epithelia, the epithelium of the cornea is maintained by stem cells. This review will discuss what is currently known about the properties of these stem cells, the clinical consequences of stem cell failure and the potential for stem cell therapy in regeneration of the ocular surface.
[Show abstract][Hide abstract] ABSTRACT: To investigate the effect of human serum, an ocular therapy, on human corneal fibroblast (HCF) wound-healing activities.
The water soluble tetrazolium reagent-1, chemotactic chambers, fibroblast-populated type-I collagen gels, zymography, and Western blotting were used to assess HCF proliferation, migration, contraction, and matrix metalloproteinase (MMP) activity and levels, respectively. Fibroblasts were obtained from human donor corneas. Human serum, fibroblast culture medium (FCM; Dulbeccos Minimal Essential Medium/10% newborn calf serum) with and without calf serum supplementation, and 0.3% hypromellose were compared.
Proliferation and migration were maximal in 1% human serum. Relaxed gel contraction was maximal for fibroblasts cultured in 10%, 50%, and 100% serum and FCM. Whereas in stressed gels, maximal contraction was induced when fibroblasts were cultured in 50% and 100% serum. In low serum concentrations, greater MMP-2 activity was detected than MMP-1 and MMP-9.
Low concentrations of human serum stimulated HCF migration, proliferation, and MMP activity. High concentrations produced greater matrix contraction. This may have implications for the therapeutic use of autologous serum.
Current eye research 09/2008; 33(8):641-52. DOI:10.1080/02713680802254790 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The corneal epithelium is continuously renewed by a population of stem cells that reside in the corneoscleral junction, otherwise known as the limbus. These limbal epithelial stem cells (LESC) are imperative for corneal maintenance with deficiencies leading to in-growth of conjunctival cells, neovascularisation of the corneal stroma and eventual corneal opacity and visual loss. One such disease that has traditionally been thought to be due to LESC deficiency is aniridia, a pan-ocular congenital eye disease due to mutations in the PAX6 gene. Corneal changes or aniridia related keratopathy (ARK) seen in aniridia are typical of LESC deficiency. However, the pathophysiology behind ARK is still ill defined, with current theories suggesting it may be caused by a deficiency in the stem cell niche and adjacent corneal stroma, with altered wound healing responses also playing a role (Ramaesh et al, International Journal of Biochemistry & Cell Biology 37:547-557, 2005) or abnormal epidermal differentiation of LESC (Li et al., The Journal of Pathology 214:9, 2008). PAX6 is considered the master control gene for the eye and is required for normal eye development with expression continuing in the adult cornea, thus inferring a role for corneal repair and regeneration (Sivak et al., Developments in Biologicals 222:41-54, 2000). Studies of models of Pax6 deficiency, such as the small eyed (sey) mouse, should help to reveal the intrinsic and extrinsic mechanisms involved in normal LESC function.
[Show abstract][Hide abstract] ABSTRACT: 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.
Prospective, noncomparative, interventional case series.
Ten eyes of 10 patients with profound LESC deficiency arising from chemical injury (4 eyes), aniridia (3 eyes), ectodermal dysplasia (1 eye), Reiger's anomaly with Pax6 haploinsufficiency (1 eye), and unknown cause (1 eye).
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.
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.
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).
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.
[Show abstract][Hide abstract] ABSTRACT: The complex mechanisms by which transforming growth factor beta (TGFbeta) regulate re-epithelialisation following injury of stratified epithelia are not fully understood. TGFbeta signals via binding to distinct receptors activating downstream effectors, including Smads which initiate transcription of target genes. However, studies have shown that TGFbeta can also signal independently of Smads through MAPK pathways, demonstrating the diversity of TGFbeta signalling. Connective tissue growth factor (CTGF) is strongly induced by and acts downstream of TGFbeta causing pathophysiology in tissues by inducing matrix deposition, conversion of fibroblasts into contractile myofibroblasts (e.g. dermis and corneal stroma) and stimulation of epithelial-to-mesenchymal transition (e.g. kidney and lung) all of which are known to cause fibrosis. However, a role for CTGF in epithelial cell function which does not involve direct contribution to fibrosis has not been demonstrated. We show for the first time that synthesis of CTGF in cultures of human corneal epithelial cells is induced by TGFbeta through the Ras/MEK/ERK MAPK signalling pathway and that this is required for re-epithelialisation to occur through cell migration. These data reveal a novel function for CTGF in the regulation of epithelial tissue repair beyond its established role in fibrosis, and further highlight the complexity of TGFbeta regulation of epithelial cell function.
Experimental Cell Research 02/2008; 314(1):131-42. DOI:10.1016/j.yexcr.2007.09.001 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ex vivo cultured limbal epithelial stem cells have been used successfully to treat corneal limbal stem cell deficiency. We identified 17 reports of the application of this novel cell-based therapy in humans. In addition we identified four reports of the use of culture oral mucosal epithelial cells to treat limbal stem cell deficiency. We examined these reports to discern the success rate, complication rate, visual outcome, whether there is an optimal technique and which patients are the most likely to benefit. We also discuss the different culture methods employed and the regulations governing cell banks that are providing this service. We found that the techniques used to cultivate and transplant cells varied, but that no individual method was clearly superior. The reported success rate is similar across all studies for both allografts and autografts. The clinical indications for this treatment are not clearly defined as indicated by the variety of disorders treated. Follow-up is limited and the long-term success rate is yet to be established. Nonetheless, we conclude that there is sufficient evidence to support the continued use and refinement of this procedure as a treatment for corneal stem cell deficiency.
Survey of Ophthalmology 09/2007; 52(5):483-502. DOI:10.1016/j.survophthal.2007.06.013 · 3.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 p63alpha 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. Disclosure of potential conflicts of interest is found at the end of this article.
[Show abstract][Hide abstract] ABSTRACT: Restoring vision in patients suffering from previously intractable blinding ocular surface disease has become possible with the advent of techniques for ex vivo expansion and transplantation of limbal epithelial stem cells onto the cornea. This approach represents one of the few adult stem cell therapies presently in the clinic. This article highlights several key research areas where progress will be made to specifically understand the biology and therapeutic potential of limbal epithelial stem cells, which may have an applicability to the understanding of other adult stem cell populations.
[Show abstract][Hide abstract] ABSTRACT: The concept of stem cell therapy has engaged the attention of the public and scientists alike. Intensive research effort is focused upon understanding the biology and therapeutic potential of embryonic and adult stem cells, with the eventual goal of treating such pathologies as Parkinson's disease, diabetes, neurological injury and degenerations and cancer. Ex vivo expansion and transplantation of limbal epithelial stem cells to the corneas to treat blinding ocular surface disease was one of the first stem cell therapies to successfully reach the clinic. However, limbal epithelial stem cell research and therapy delivery has remained largely within the noncommercial academic clinician-scientist environment from which it was originally pioneered. In our experience, gaining regulatory approval has been as great a hurdle as surmounting the scientific challenges of stem cell therapy. Based upon our model of delivering 'accredited' limbal epithelial stem cell therapy to patients in compliance with Good Manufacturing Practice and the new European Union Tissues and Cells Directive, we address the key regulatory questions. This may help colleagues who are developing innovative academic research-driven stem cell therapies regarding donor consent, raw materials, quality assurance, laboratory specification, indemnity and funding.
Regenerative Medicine 10/2006; 1(5):715-9. DOI:10.2217/174607184.108.40.2065 · 2.79 Impact Factor