Bjørn Nicolaissen

Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement.

In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.

A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain.

Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes (n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy (n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10-30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-beta receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human CB spheres are vital to fully utilize them as a clinical source of retinal progenitor cells in the future.

Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.

A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2×2×0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.

A novel method for preserving cultured limbal epithelial cells

Aim: To investigate organ culture preservation of cultured limbal epithelial cells in order to enhance the availability of tissue-engineered epithelia that are used to treat patients with limbal stem cell deficiency. Methods: Limbal epithelial cells were cultured for 3 weeks on intact amniotic membrane fastened to a polyester membrane carrier. The cultured epithelia were stored for 1 week at 23°C in organ culture medium. The preserved epithelia were then examined using a colorimetric cell viability assay, light microscopy and immunohistochemistry. Results: The viability of the preserved epithelia was 84% (20%), and no statistically significant difference was found compared with non-preserved epithelia. In general, the cell borders were maintained, the nuclei showed no sign of degeneration, and the original layered structure was preserved. Mild intercellular oedema was occasionally observed. Expression of p63, K19 and vimentin was maintained. Conclusions: Cultured limbal epithelial cells can be preserved in organ culture medium for 1 week at room temperature, while maintaining the original layered structure and undifferentiated phenotype.

Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series

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.

DNA damage in lens epithelium of cataract patients in vivo and ex vivo

Purpose:  DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system.

Corneal collagen crosslinking in vitro: Inhibited regeneration of human limbal epithelial cells after riboflavin–ultraviolet-A exposure

PURPOSE: To assess the hypothesis that during corneal crosslinking (CXL) treatment, riboflavin and ultraviolet‐A (UVA) may have a toxic effect on human limbal epithelial cells. SETTING: Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway. DESIGN: Experimental study. METHODS: In this vitro study, limbal biopsies from corneoscleral rims collected after corneal transplantation were treated with the following combinations: riboflavin–UVA, riboflavin only, or UVA only; a control group received no treatment. After 3 weeks of cell culture, outgrowth of epithelium from the biopsies was evaluated by measuring the area of cell expansion and the number of cell layers. The explanted biopsies were analyzed for proliferation using immunohistochemistry marker Ki‐67 and for apoptosis using the terminal deoxynucleotidyl transferase deoxy‐UTP‐nick end labeling (TUNEL) assay. RESULTS: The mean outgrowth from the biopsies was 2.25 mm2 ± 6.90 (SD) in the riboflavin–UVA group, 181.4 ± 94.8 mm2 in the riboflavin‐only group, 128.5 ± 129.5 mm2 in the UVA‐only group, and 176.2 ± 114.0 mm2 in the control group. There were no statistically significant between‐group differences in the number of cell layers except in the riboflavin–UVA group, in which no cells were found. Detection of apoptosis with the TUNEL‐assay was found in the riboflavin–UVA group only (4/5 sections). The proliferation marker Ki‐67 was positive in some sections in all groups. CONCLUSION: Cytotoxicity and reduced cell expansion of human limbal epithelial cells occurred after riboflavin–UVA treatment in vitro, emphasizing the importance of avoiding riboflavin–UVA on the limbus during CXL. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Regeneration with proliferation of the endothelium of cultured human donor corneas with extended postmortem time.

Purpose: To examine the endothelium of donor corneas with extended postmortem time for survival and reparative mechanisms in an eye bank organ culture storage system. Methods: We obtained 14 pairs of donor corneas with a postmortem time ranging from 29 to 163 hours. One cornea of a pair was immediately fixed for the study of structural changes postmortem and to serve as a control. The second was stored in organ culture for 3 days and thereafter fixed to be studied for reparative processes. Examination was done with light microscopy and scanning electron microscopy. Immunohistochemical staining with antibodies against proliferating cell nuclear antigen, Ki-67, and n-cadherin was performed to examine for cell proliferation and to characterize the cells. Results: The control corneas showed increasing endothelial cell damage with increasing postmortem time. After 5-7 days postmortem, most cells were structurally damaged. After 3 days in organ culture, all corneas acquired an endothelial covering of the posterior surface, with cells, suggesting proliferation in both scanning preparations and in cross-sections. Positive endothelial cell staining with proliferating cell nuclear antigen was found in all cultured corneas. Ki-67 staining of the endothelium was found in 9 of the cultured corneas. Conclusions: The study showed survival of the corneal endothelium up to 7 days postmortem, and accordingly, the potential clinical use of donor corneas with extended postmortem time. Our results furthermore suggest that repair of the endothelium in donor corneas during organ culture storage occurs also by proliferation and not only by migration and enlargement of existing cells. If we uncover the mechanisms regulating cell proliferation in corneal endothelium, it should be possible to develop better storage methods of corneal transplants to improve quality and supply.

Topical anesthesia and adjustable sutures in strabismus surgery.

Abstract Our experience with the use of topical anesthesia and adjustable sutures in a one‐stage operation for treatment of strabismus is discussed, and selected cases are presented. In addition to using the method in recession, we have also developed a method for using adjustable sutures in resection of muscles. We have also found the method useful in reoperations and in operations on paretic muscles.

Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy.

In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial - and NSC-associated markers both after direct fixation and repetitive passages. In conclusion, the adult human eye may harbor two different populations of neuroepithelial stem/progenitor cells; a non-glial population located in the proximal pars plana around peripheral cysts in addition to a population with Müller glia characteristics. Yet, we only found that the glial population was able to respond to retinal injury by targeted migration into the vitreous.