Limbal stem cells maintain the corneal epithelium through a process of clonal growth and ordered migration. In X-inactivation mosaic female mice, that express LacZ from one of their X-chromosomes, random clumps of LacZ-positive cells are seen in the cornea at 3-6 weeks of life. This pattern resolves between 6-10 weeks forming radial stripes thought to represent chords of clonally related, inwardly migrating cells. By measuring the number and width of stripes and correcting for the effects of different proportions of LacZ-positive cells, an estimate of the number of coherent stem cell clones maintaining the tissue can be derived. Analysis at 5 ages demonstrated that the estimated number of coherent stem cell clones is reduced from ~100 at 15 weeks to ~50 at 39 weeks and is then stable at least until 52 weeks. An automated method was developed using image analysis software to analyse these striping patterns. This method produced results that did not differ significantly from the above. The dosage of the transcription factor Pax6 is crucial for normal eye development. In Pax6 heterozygous animals the estimated number of coherent stem cell clones is reduced to ~50 at 15 weeks with no further reduction up to 30 weeks. Mice hemizygous for the PAX77 transgene over-express human PAX6. In PAX77 hemizygous X-inactivation mosaics, estimated clone number was similarly reduced to ~50 with no further decline. Mice heterozygous for both Gli3 and Pax6 have a distinct striping phenotype, highlighted by an increase in coherent clones. When the corneal epithelium is injured the surrounding epithelial cells migrate along the corneal stroma to cover the wound. X-gal staining of healed, centrally wounded X-inactivation eyes reveals that striping patterns are reconstituted during wound healing in ex-vivo culture. In GFP mosaics the healing process can be imaged using time-lapse confocal microscopy. This technique demonstrated that clones remain contiguous throughout their migration. Healing of peripheral wounds was observed to form de-novo whorling patterns, revealing that basal cells in the epithelium can migrate both away from and towards the limbal region.

Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...

Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...

yes / The cornea comprises a pool of self‐regenerating epithelial cells that are crucial to preserving clarity and visibility. Limbal epithelial stem cells (LESCs), which live in a specialized stem cell niche (SCN), are crucial for the survival of the human corneal epithelium. They live at the bottom of the limbal crypts, in a physically enclosed microenvironment with a number of neighboring niche cells. Scientists also simplified features of these diverse microenvironments for more analysis in situ by designing and recreating features of different SCNs. Recent methods for regenerating the corneal epithelium after serious trauma, including burns and allergic assaults, focus mainly on regenerating the LESCs. Mesenchymal stem cells, which can transform into self‐renewing and skeletal tissues, hold immense interest in tissue engineering and innovative medicinal exploration. This review summarizes all types of LESCs, identity and location of the human epithelial stem cells (HESCs), reconstruction of LSCN, and artificial stem cells for self‐renewal.

The conventional view for corneal epithelial maintenance suggests that a stem cell population found in the limbus (at the rim of the cornea) produces daughter cells, called transient amplifying cells, which migrate centripetally. This limbal stem cell (LSC) hypothesis was recently questioned and the alternative model suggests that stem cells are present throughout the corneal epithelium. The main aims of this thesis were to investigate whether age and Pax6 genotype affect LSC function. Previous work with X-inactivation mosaics revealed radial stripes of β-galactosidase-expressing cells in the corneal epithelium (from about 5 weeks of age), which decreased with age and were reduced in Pax6+/- mice (a model for aniridia, a human eye disease). The reduction in Pax6+/- mice could be due to either reduced LSCs function or a more coarse-grained mosaicism caused by reduced cell mixing during development. Comparison of patch sizes in Pax6+/- and wild-type X-inactivation mosaics showed that patches were smaller in Pax6+/- cornea epithelia before the initiation of stripes (3 weeks of age). This implies that stripe-number reduction is not caused by reduced cell mixing, so an effect on LSC function remained a possibility. Thus, the numbers of label-retaining cells (putative stem cells) in Pax6+/- were compared to controls at 15 and 30 weeks old but they were not reduced at 30 weeks or in Pax6+/- mice, as had been predicted. The failure to demonstrate the predicted result suggests either that the hypothesis was incorrect or the experimental approach was inappropriate. Furthermore, it was discovered that mice expressing β-galactosidase under the keratin 5 promoter produced rare stripes in the corneal epithelium, which are likely to represent clonal lineages derived from individual stem cells. Older mice demonstrated a significantly lower frequency of stripes, a result compatible with the predicted reduction of active LSC with age. Pax6+/- corneas were highly abnormal and stripes were not formed properly, so direct comparison was not possible. Finally, pilot experiments with conditional expression of a reporter gene revealed the successful formation of a stripe, and hence provide a plausible alternative approach to compare stripe numbers reflecting active LSCs but the method has yet to be optimised. Overall, the results suggest that LSCs are reduced with age and support the limbal location of stem cells maintaining the corneal epithelium.

The limbal stem cell population is located in the limbal junctional zone between the cornea and the conjunctiva, and is responsible for maintaining the corneal epithelium. Damage to the limbal stem cell population results in a condition known as limbal stem cell deficiency (LSCD), which is characterised by conjunctivalisation of the cornea, visual impairment and persistent irritation. To treat LSCD, an alternative source of human limbal epithelial (HLE) cells must be transplanted back onto the diseased cornea. Limbal tissue grafts have had a moderate degree of success. However, autologous grafts risk damage to the healthy eye, whilst allogeneic grafts are susceptible to immunological rejection. Cultured HLE grafts offer a promising alternative to whole tissue grafts. The production of cultured HLE grafts involves the removal of a small (1-2 mm2) biopsy from the patient’s healthy limbus, followed by ex vivo expansion to produce an epithelial sheet, which is subsequently transplanted onto the damaged corneal surface. However, the production of cultured HLE grafts usually requires the addition of animal-derived products during cell culture. Animal-derived components, such as foetal bovine serum (FBS) and murine 3T3 feeder cells, introduce the patient to potential crossspecies infection and immune responses to xenogeneic antigens. Consequently, the overall aim of this project has been to develop a culture technique free of xenogeneic products for the establishment and propagation of HLE cells. To achieve this aim, alternatives to FBS in the culture medium and 3T3 feeder cells were pursued. A defined serum-free medium (SFM) containing vitronectin (VN), insulin-like growth factor binding protein 3 (IGFBP3), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) was investigated as an alternative to serumsupplemented medium (SSM) for HLE cell culture. Initial studies focused on the effects of these growth factors on HLE cell metabolic activity and migration. Metabolic activity was primarily stimulated by IGF-I and EGF, with the combination of IGF-I and EGF in solution stimulating metabolic activity to a significantly greater extent than the SSM positive control (p = 0.006). HLE cell migration was also effected by combinations of VN, IGFBP3, IGF-I and EGF. Migration was stimulated above the SFM negative control by the combination of IGFBP3 and IGF-I either with or without the addition of EGF. However, the presence of VN was required for optimal migratory responses (p < 0.003). Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) were also investigated as additional components to the SFM formulation. HGF significantly stimulated HLE cell metabolic activity and migration (p < 0.02). In contrast, KGF did not significantly stimulate either HLE cell metabolic activity or migration. The addition of either HGF or KGF to the SFM supplemented with VN, IGFBP3, IGF-I and EGF did not significantly enhance the metabolic activity of HLE cells. Therefore, HGF and KGF were no longer pursued as additional components to the SFM formulation. Additional studies were conducted to examine the efficacy of replacing murine 3T3 feeder cells with human ocular stromal cells during HLE cell culture. Initially, stromal cells were isolated from the cornea, limbus and sclera to determine whether there were differences between these stromal cell populations. The results indicated that scleral stromal cells had a significantly larger area and perimeter than either corneal or limbal stromal cells (p < 0.001). Scleral stromal cells were also significantly more rounded than either corneal or limbal stromal cells, as determined by the elliptical factor equation (p < 0.001). Immunocytochemistry also revealed that scleral stromal cells expressed significantly more of the myofibroblast marker ..- smooth muscle actin than either corneal or limbal stromal cells (p < 0.001), and significantly less of the fibroblast/myofibroblast marker Thy-1 than corneal or limbal stromal cells (p < 0.001). Therefore, scleral stromal cells were identified as different in comparison to corneal and limbal stromal cells. Primary HLE cells were cultured with irradiated corneal, limbal and scleral stromal cells. HLE cultures established with either corneal or limbal stromal feeder cells contained more cellular protein (as measured by rhodamine B dye absorbance) than cultures established without feeder cells (p < 0.001). The colony forming efficiency (CFE) of HLE cells established with corneal or limbal stromal feeder cells was also significantly greater than HLE cells established without feeder cells (p < 0.001). In contrast, HLE cultures established with scleral stromal feeder cells contained low levels of cellular protein and had a low CFE, which was not significantly different to the HLE cultures established without feeder cells. Immunocytochemistry indicated that HLE cultures established with scleral feeder cells also showed lower expression of the stem cell markers ABCG2 and C/EBP ... These results suggest that freshly isolated HLE cells can be cultured with irradiated corneal or limbal stromal cells as a replacement for murine 3T3 feeder cells. Finally, the SFM supplemented with VN+IGFBP3+IGF-I+EGF was combined with limbal stromal feeder cells, and examined as a culture technique free of animalderived products. Freshly isolated HLE cells established in SFM supplemented with VN+IGFBP3+IGF-I+EGF and limbal feeder cells contained a similar amount of cellular protein (as measured by crystal violet dye absorbance) when compared to the SSM+3T3 positive control. In addition, the CFE of freshly isolated HLE cells established in VN+IGFBP3+IGF-I+EGF and limbal feeder cells was significantly higher than the SSM+3T3 positive control (p = 0.004). However, a live/dead assay revealed a reduced HLE cell viability in SFM supplemented with VN+IGFBP3+IGFI+ EGF and limbal feeder cells after seven days in culture. In addition, immunocytochemistry demonstrated a lower expression of the stem cell markers ABCG2 and C/EBP .. in the SFM treatment with limbal feeder cells. Therefore, freshly isolated HLE cells can be cultured in SFM supplemented with VN+IGFBP3 +IGF-I+EGF and limbal feeder cells. However, this culture technique is less likely to support the growth of immature limbal stem cells when compared to the SSM+3T3 positive control. Overall, this research has attempted to create a culture system free of animal-derived products for the production of cultured HLE grafts to treat limbal stem cell deficiency. The results show that HLE cells respond to a serum-free medium formulation containing VN+IGFBP3+IGF-I+EGF. In addition, this culture medium can be combined with irradiated stromal cells isolated from the limbus to support HLE culture production. However, the combination of VN+IGFBP3+IGF-I+EGF and limbal feeder cells demonstrated a reduced viability, which indicates that further refinement of the formulation is required. This thesis has also demonstrated differences between stromal cells isolated from the cornea, limbus, and sclera, and has generated knowledge which may impact on the understanding of stromalepithelial regulation.

yes / We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

No / The aim of this study was to develop a synthetic hydrogel to act as a corneal substitute capable of selectively supporting the adhesion and proliferation of limbal epithelial cells (LECs) while inhibiting growth of limbal fibroblasts. Deficiency of LECs causes conjunctival epithelial cells to move over the cornea, producing a thick scar pannus. Unilateral defects can be treated using LEC cultured from the unaffected eye, transplanting them to the affected cornea after scar tissue is removed. The underlying wound bed is often damaged, however, hence the need to develop a corneal inlay to aid in corneal re-epithelialization. Transparent epoxy-functional polymethacrylate networks were synthesized using a combination of glycerol monomethacrylate, ethylene glycol dimethacrylate, lauryl methacrylate and glycidyl methacrylate that produced two different bulk hydrogel compositions with different equilibrium water contents (EWCs): Base 1 and Base 2, EWC=55% and 35%, respectively. Two sets of amine-functional hydrogels were produced following reaction of the epoxide groups with excesses of either ammonia, 1,2-diamino ethane, 1,3-diamino propane, 1,4-diamino butane or 1,6-diamino hexane. Neither series of hydrogels supported the proliferation of limbal fibroblasts irrespective of amine functionalization but they both supported the adhesion and proliferation of limbal epithelial cells, particularly when functionalized with 1,4-diamino butane. With Base 1 hydrogels (less so with Base 2) a vigorous epithelial outgrowth was seen from small limbal explants and a confluent epithelial layer was achieved in vitro within 6days. The data support the development of hydrogels specific for epithelial formation.

Yes / The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variabil-ity and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmis-sion, we sought to determine if decellularisation and/or c-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without c-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, DNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and c-irradiation did not significantly affect the LSC phe-notype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p < 0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a c-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer.

The damage of ocular surface represents one of the most common causes of decreased quality of vision or even blindness. If the injury is extensive and includes the region of limbus, niche of limbal stem cells (LSC), LSC deficiency occurs and the natural corneal regeneration is stopped. Conjunctival epithelium migrates into the injured area. Neovascularization, local inflammation and corneal opacity occur. Corneal transplantation is an insufficient treatment in such case. If the injury is bilateral, the allogenic limbal graft or LSC transplantation is required. In such cases systemic immunosuppressive drugs with many negative side-effects must be administered. The search for an adequate autologous substitution is important for avoid immunosuppressive medication. Mesenchymal stem cells (MSC) represent a perspective substitution for the reason of their immunomodulatory properties and the capability to differentiate in many cell types. There is possibility to isolate sufficient number of these cells from adipose tissue or bone marrow which are relatively easily accessible. Our goal was to observe local production of cytokines and other molecules which are present in inflammatory reaction after the chemical burn of the murine cornea and after the treatment with stem cells growing on nanofiber scaffold....