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Purpose Electrospun polymer nanofibers present a structure similar to extracellular matrix (ECM) and support the surrounding cells. The type of polymer used, as well as the polymer blending-ratio, affects wettability. This in turn influences cell growth by altering the types of proteins adsorbed from the solution. Human corneal epithelial cells were grown on electrospun biodegradable and non-biodegradable polymer membranes to evaluate the membranes’ potential within ocular surface tissue engineering. Methods The electrospun polymer membranes consisted of cellulose acetate, poly (lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), and blends of PCL and polyethylene glycol (PEG). Expression of CDH1 (E-cadherin), ITGA6, ITGB4 (Integrin α6β4) and MKI67 (Ki67) genes were measured in all conditions after 14 days of culture using qRT-PCR. Results Cells seeded on all types of membranes expressed MKI67, which is associated with cell cycle activity and correlates with cell growth. MKI67 expression was high in cells grown on 3:2 blends of PCL and PEG (2.88) and PCL (2.75). This amount was 1.75, 1.00 and 1.59 for cells grown on cellulose acetate, 1:1 blend of PCL and PEG and PLGA respectively. The amount of ITGA6, ITGB4 expression was 1.72 in cells grown on PCL membranes, 1.10 for 3:2 blend of PCL and PEG, 1.05 for cellulose acetate, 1.53 for PLGA and 1.01 for 1:1 blend of PCL and PEG. Only cells grown on membranes containing blends of PCL: PEG showed CDH1 expression, which is critical for cell-cell contact. Conclusions All polymer membranes enabled cellular adhesion and proliferation. PCL:PEG and PCL membranes appear to promote the highest proliferative activity and strongest cell-membrane/cell-cell adhesion. Thus, these might be the most promising substrates for epithelial ocular surface tissue engineering.

ummary The anatomical and biological properties of stem cells in the eye have been the topic of indepth research over the last few decades. Enormous advances in the cell and tissue isolation and cultivation techniques, as well as molecular characterization of the different cell populations have been achieved, next to the major advances made in the bioscaffolds’ engineering for cell delivery and treatment of eye diseases. Advances in the cell and gene therapy have reached culmination with the human cornea being at the top and holding great promise in treating eye disorders. The eye is indeed a golden mine for stem cells. This session will include lectures from European experts in the field with high international recognition and large collaborative networks. Challenges in the clinical applications of cornea limbal stem cells will be described, along with advances in corneal endothelium engineering for future transplantation applications. In addition, examples of regulating the gene expression towards solving ocular surface diseases will be shown, as well as future directions hallmarking stem cell and cell therapy in ophthalmology.

Purpose Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. Methods We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. Results Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up). Conclusions Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.

Purpose p63 (isoform ΔNp63α) is a putative stemness marker for limbal epithelial stem cells (LESCs), and is essential for the proliferative potential of stem cells in stratified epithelia. The niche for LESCs is the limbus, located between the clear cornea and bulbar conjunctiva. LESCs are responsible for and able to renew and repair the corneal epithelium throughout life. In a number of disorders and after trauma, limbal stem cell deficiency (LSCD) may occur, and this can be treated by isolation and cultivation of LESCs from a healthy eye followed by transplantation to the affected eye. It is known that the percentage of p63-bright cells affect the success rate of the transplantation. Thus, we wanted to reveal whether lithium, a known Wnt signaling inducing agent, may increase the expression of p63 in human LESC cultures. Methods Small limbal biopsies (1x1-2mm) were dissected from donor corneas and cultured in a standard medium at 37 oC and 5% CO2 under different concentrations of lithium (LiCl2). The cells were cultured for 3-6 weeks and medium was changed every 2-3 days. Results The epithelial cells migrated from the limbal biopsy to form an epithelial sheet. Lithium enhanced the expression of p63 compared to controls, and this was confirmed by qRTPCR. Further studies mapping epigenetic alterations of the lithium treatment are currently conducted. Conclusion Lithium induced upregulation of p63 in LESC could be clinically important by increasing the long term success of limbal stem cell therapy.

Purpose: Storage time for donor corneas in Optisol GS is limited compared to Eye Bank Organ Culture (EBOC). We here examine the epithelium on donor corneoscleral rims after primary storage in Optisol GS and subsequent incubation in EBOC. Methods: Morphology was monitored by light and electron microscopy, expression of phenotypic and genotypic markers by immunohistochemistry and RT-PCR and changes in oxidative lipid and DNA damage by ELISA and COMET assay. Results: A prominent loss of cells was observed after storage in Optisol GS. After maintenance in EBOC, spreading apical cells were Occludin+, while the staining for E-cadherin and Connexin-43 was less intense. There were an upregulation of Occludin and a downregulation of E-cadherin and Connexin-43. Eye Bank Organ Culture was associated with an ongoing proliferative activity and a downregulation of putative progenitor/stem cell marker ABCG2 and p63. Staining for 8-OHdG and Caspase-3 did not increase, while levels of malondialdehyde and number of DNA strand breaks and oxidized bases increased. Conclusions: This dual procedure should be pursued as an option to increase the storage time and the pool of available donor corneas. The observed downregulation of markers associated with stemness during EBOC is relevant considering the potential use of donor epithelium in the treatment of ocular surface disorders. Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#\OnlineOpen_Terms