Paracrine activity of adipose derived stem cells on limbal epithelial stem cells.

Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of patients for the cornea transplantation all over the world and the list is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient's body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and has a potential role in mitigation of the adverse impact of inflammation induced by lipopolysaccharide.

A decellularized human corneal scaffold for anterior corneal surface reconstruction.

Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium.

Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.

Keratin expression by corneal and limbal stem cells during development.

Keratins are the forming units of intermediate filaments (IF) that provide mechanical support, and formation of desmosomes between cells and hemi desmosomes with basement membranes for epithelium integrity. Keratin IF are polymers of obligate heterodimer consisting one type I keratin and one type II keratin molecules. There are 54 functional keratin genes in human genome, which are classified into three major groups, i.e., epithelial keratins, hair follicle cell-specific epithelial keratins and hair keratins. Their expression is cell type-specific and developmentally regulated. Corneal epithelium expresses a subgroup of keratins similar to those of epidermal epithelium. Limbal basal stem cells express K5/K14, and K8/K18 and K8/K19 IF suggesting that there probably are two populations of limbal stem cells (LSCs). In human, LSCs at limbal basal layer can directly stratify and differentiate to limbal suprabasal cells that express K3/K12 IF, or centripetally migrate then differentiate to corneal basal transient amplifying cells (TAC) that co-express both K3/K12 and K5/K14 prior to moving upward and assuming suprabasal cells phenotype of only K3/K12 expression that signifies corneal type epithelium differentiation. In rodent, the differentiated cornea epithelial cells express K5/K12 in lieu of K3/K12, because K3 allele exists as a pseudogene and does not encode a functional K3 protein. The basal corneal cells of new-born mice originate from surface ectoderm during embryonic development slowly commit to differentiation of becoming TAC co-expressing K5/K12 and K5/K14 IF. However, the centripetal migration may still occur at a slower rate in young mice, which is accelerated during wound healing. In this review, we will discuss and compare the cornea-specific keratins expression patterns between corneal and epidermal epithelial cells during mouse development, and between human and mouse during development and homeostasis in adult, and pathology caused by a mutation of keratins.

Primary Culture of Cornea-Limbal Epithelial Cells In Vitro.

The cultivation of corneal-limbal cells in vitro represents an excellent means to generate models to study cornea function and disease processes. These in vitro expanded cornea-limbal epithelial cell cultures are rich in stem cells for cornea, and hence can be used as a cell therapy for cornea-limbal deficiency. This chapter details the primary culture of these cornea-limbal cells, which can be used as model for further studies of the cornea surface.

Optimization of Human Limbal Stem Cell Culture by Replating a Single Limbal Explant.

Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.

Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants.

To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]-P2). Cells were characterized by alcian blue/periodic acid-Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0-P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.

Comparative study of substrate free and amniotic membrane scaffolds for cultivation of limbal epithelial sheet.

Transplantation of cultivated limbal epithelial transplantation has been proven to restore the corneal surface in limbal stem cell deficiency (LSCD). Here we comparatively investigated the optimized conditions and the efficiency of limbal epithelial sheet growth in three media conditions as well as with substrate free (transwell), human amniotic membrane (HAM) sutured onto transwell inserts (HAMTW), and HAM slide scaffold (HAMS). Outcomes evaluated were outgrowth sheet size from limbal explants, expression of stem/progenitor cell markers p63α, ABCG2 and CK15, and colony formation efficiency (CFE). Additionally, limbal epithelial sheets on HAMS were transplanted into corneas of LSCD rabbit models. Limbal epithelial sheets with 5% human AB serum showed the greatest increase in ABCG2 efflux activity (JC1low), p63α expression, and CFE compared in both conditions without HAM and with HAM, respectively. The outgrowth sheet size, cell yield, and Ki67 expression were increased in limbal epithelial sheets on HAMS compared to transwell and HAMTW. ABCG2 efflux activity, p63α and CK15 expressions, and CFE were also increased in limbal epithelial sheets on HAMS as well. In corneas of transplanted rabbit LSCD models, p63α expressions were noted in the basal layers and CK12 expressions were observed in superficial layers. Cultivation of limbal epithelial sheet on HAMS with xeno-free medium enhances the growth and stemness of limbal epithelial sheets.

Morphology of the extraocular muscles (m. bulbi) in the pre-hatchling and post-hatchling african black ostriches (struthio camelus domesticus L., 1758) (Aves: Struthioniformes).

The aim of the study was to describe the morphology and the development of the extraocular muscles (EOMs) in the pre-hatchling and post-hatchling African black ostrich. The study involved 50 birds aged between 28 days and 3 years. The EOMs were analyzed morphologically with respect to the location and length of the straight and oblique muscles and the third eyelid muscles, the length and breadth of their tendons as well as the distance and shape of the muscle tendon insertions at the corneal limbus. A histological and histometric analysis were also carried out. The greatest increase in the length of the EOMs was noted in groups III-V. A marked increase in the length of the tendons of the dorsal straight muscle was found in groups II and III, in the tendons of the nasal straight muscle in groups IV and V, in the tendons of the dorsal oblique muscle in groups III to V and in the tendons of the ventral oblique muscle in groups IV and V. There was a significant increase in the breadth of the dorsal straight and ventral oblique muscle tendons in groups IV and V and the tendons of the pyramidal muscle in groups III and V. The distance of the distal insertion of the tendon at the corneal limbus increased steadily with age in all the examined groups. The number of fascicles and muscle fibres, their diameter and length in all the studied EOMs were different in the different groups.

In Vitro Compatibility of Contact Lenses With Corneal Epithelial Cells.

PURPOSE: To determine the interaction of contact lenses of different materials with corneal epithelial cells grown in tissue culture. METHODS: Two different corneal epithelial cell lines were grown to confluence in culture media. Two hydrogel contact lenses with and without polyvinylpyrrolidone (PVP) {1-DAY ACUVUE MOIST (1-Day ACUVUE [hydrogel lenses]) and a silicone hydrogel contact lens, AIR OPTIX NIGHT & DAY} were removed from their blister packs, washed in phosphate-buffered saline, and applied to the cells. After exposure for 24 hr at 37°C, lenses were removed, and the corneal cells and supernatants processed. Supernatants from the cell assays were used to quantify the amount of 17 different cytokines that were produced using a multiplex bead assay. Cells were stained to assess amount of cell death (apoptosis or necrosis) or stained to determine the level of mitochondrial activity. Stimulants of necrotic death (latex) or apoptotic death (sorbitol) were used as positive controls. RESULTS: Cells produced cytokines during normal growth. Exposure of cells to the hydrogel lenses resulted in only minimal changes to normal production of cytokines, but latex or sorbitol produced the most change. Exposure of the cells to all three lenses caused 4% to 23% reduction in mitochondrial activity, whereas exposure to the positive controls caused 71% to 98% reduction in mitochondrial activity. Exposure of the corneal epithelial cells to contact lenses produced minimal morphological changes, whereas exposure to latex or sorbitol produced significant changes to the human corneal epithelial cell line. CONCLUSIONS: Exposure of corneal epithelial cells to contact lenses had minimal impact on their physiology. There was no difference in epithelial cell responses to hydrogel with or without PVP compared with the silicone hydrogel contact lens.

Human Umbilical Cord Lining Cells as Novel Feeder Layer for Ex Vivo Cultivation of Limbal Epithelial Cells.

PURPOSE: To determine the effectiveness of human umbilical cord-derived mucin-expressing cord lining epithelial cells (CLEC-muc) as feeder cells in a coculture system for the cultivation of human limbal stem cells. METHODS: Human CLEC-muc were cultured in PTTe-1 medium and treated with mitomycin C to arrest their growth to make the feeder layer. Single-cell suspension of limbal cells was prepared from corneal rim collected from the Singapore Eye Bank. Limbal cells were cultured in a coculture system with CLEC-muc as well as 3T3 cells as feeder layer. We compared the colony-forming efficiency and cell morphology of the limbal cells cultured in the two different feeder layers. We also compared the expression level of several putative limbal stem cell markers, such as HES1, ABCG2, ΔNP63, and BMI1, in the cultured limbal cells by immunostaining and quantitative (q)RT-PCR. Expression of cytokeratins CK14, CK15, CK19, CK3, and CK4 was further compared. RESULTS: Human limbal epithelial cells cultured in both types of feeder layers showed comparable cell morphology and colony-forming efficiency. These cells exhibited a similar expression pattern of HES1, ABCG2, ΔNP63, BMI1, CK14, CK15, CK19, and CK3 as detected by immunostaining and PCR. CONCLUSIONS: Human CLEC-muc may be a suitable alternative to conventional mouse 3T3 feeder cells, which may reduce the risk of zoonotic infection.

An investigation of donor and culture parameters which influence epithelial outgrowths from cultured human cadaveric limbal explants.

Limbal stem cell deficiency is a blinding disease which affects the cornea at the front of the eye. The definitive cure involves replacing the corneal epithelial (limbal) stem cells, for example by transplanting cultured limbal epithelial cells. One method of performing cultures is to grow a sheet of epithelial cells from a limbal explant on human amniotic membrane. The growth of limbal tissue can be variable. The aim of this study is to investigate how different donor and culture factors influence the ex vivo growth of cadaveric limbal explants. Limbal explant cultures were established from 10 different cadaveric organ cultured corneo-scleral discs. The growth rate and the time taken for growth to be established were determined. Statistical analysis was performed to assess correlation between these factors and donor variables including donor age, sex, time from donor death to enucleation, time from enucleation to organ culture storage and duration in organ culture. Growth curves consistently showed a lag phase followed by a steeper linear growth phase. Donor age, time between death and enucleation, and time between enucleation and organ culture were not correlated to the lag time or the growth rate. Time in organ culture had a significant correlation with the duration of lag time (P = 0.003), but no relationship with the linear growth rate. This study shows that an important factor correlating with growth variation is the duration of corneo-scleral tissue in organ culture. Interestingly, donor age was not correlated with limbal explant growth.

Culture of limbal stem cells on human amniotic membrane.

Limbal stem cells (LSC) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell (HLECs) transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. A major challenge is the ability to identify LSC in vitro and in situ, and one of the major controversies in the field relates to reliable LSC markers. This study was carried out to evaluate the culture of a limbal biopsy on human amniotic membrane (HAM): directly on the chorionic side and on intact epithelium, and the expression of the stem cell associated markers: ABCG2, p63. HAM has been extensively used for ocular surface reconstruction and has properties which facilitate the growth of epithelial cells controlling inflammation and scarring.

Discontinuous LYVE-1 expression in corneal limbal lymphatics: dual function as microvalves and immunological hot spots.

LYVE-1(+) corneal lymphatics contribute to drainage and immunity. LYVE-1 is widely accepted as the most reliable lymphatic marker because of its continuous expression in lymphatic endothelium. LYVE-1 expression in corneal lymphatics has not been examined. In this study, we report intact CD31(+) corneal lymphatic capillary endothelial cells that do not express LYVE-1. The number of LYVE-1(-) gaps initially increased until 8 wk of age but was significantly reduced in aged mice. C57BL/6 mice showed a notably higher number of the LYVE-1(-)/CD31(+) lymphatic regions than BALB/c mice, which suggests a genetic predisposition for this histological feature. The LYVE-1(-) lymphatic gaps expressed podoplanin and VE-cadherin but not αSMA or FOXC2. Interestingly, the number of LYVE-1(-) gaps in FGF-2, but not VEGF-A, implanted corneas was significantly lower than in untreated corneas. Over 70% of the CD45(+) leukocytes were found in the proximity of the LYVE-1(-) gaps. Using a novel in vivo imaging technique for visualization of leukocyte migration into and out of corneal stroma, we showed reentry of extravasated leukocytes from angiogenic vessels into newly grown corneal lymphatics. This process was inhibited by VE-cadherin blockade. To date, existence of lymphatic valves in cornea is unknown. Electron microscopy showed overlapping lymphatic endothelial ends, reminiscent of microvalves in corneal lymphatics. This work introduces a novel corneal endothelial lymphatic phenotype that lacks LYVE-1. LYVE-1(-) lymphatic endothelium could serve as microvalves, supporting unidirectional flow, as well as immunological hot spots that facilitate reentry of stromal macropahges.

Proliferative and migratory aptitude in pterygium.

Pterygium is a chronic fibrovascular overgrowth on the corneal surface and is often associated with inflammation, astigmatism and obstructed vision. The common treatment is surgical removal but post-operative recurrences with more aggressive behavior are common. However, there is a controversy in the pathogenesis of primary pterygium between limbal stem cell failure versus proliferation. In this study, we explore the proliferative and migratory aptitude in pterygium by characterizing the growth and migration pattern of pterygial cells in the head (on the cornea), the neck (over the focal limbus), and the body (on the conjunctiva) epithelia of 12 full-length primary pterygia. Immunofluorescence and quantification analyses showed a spatial expression pattern of markers for stem cells, cell growth, and matrix metalloproteinases. Beside the basal epithelia in all three regions, p63α(strong) cells were located in suprabasal layers in head, weak in the body and absent in neck. Pertinent cell proliferation in head than body epithelia was revealed by its higher colony-forming efficiency. ATP-binding cassette transporter glycoprotein family member-2 and cytokeratin-15 were found mainly in the body basal epithelia, similar to that in normal conjunctiva. Much fewer proliferating stem-like cells in the neck region supported the limbal failure as a cause of pterygium formation. Pax6, matrix metalloproteinase-2 and -9 were more expressed in the head than in the other two regions. Our results indicate the importance of pterygium head in tissue growth and invasion and its likely involvement in post-operation recurrence.

Regeneración de la superficie ocular: stem cells/células madre y técnicas reconstructivas / Regeneration of the ocular surface: stem cells and reconstructive techniques

La córnea es un tejido transparente constituido microscópicamente por 5 capas bien diferenciadas. El epitelio corneal es esencial para la transparencia corneal y se encuentra en continua renovación a lo largo de la vida a partir de la población de células madre limbocorneales. La localización de estas células madre limbocorneales parece residir en las capas basales del epitelio limbocorneal, de vital importancia para mantener el microambiente de estas células madre limbocorneales, que depende de una variedad de factores intrínsecos y extrínsecos. La insuficiencia límbica se produce cuando ocurre una pérdida parcial o total de estas células madre limbocorneales. Este cuadro lleva a una opacificación corneal con la consiguiente pérdida de visión. En estos casos, el trasplante corneal supone únicamente un reemplazo temporal del epitelio corneal; es necesario llevar a cabo un tratamiento previo con trasplante de limbo autólogo o alogénico, que permita regenerar la población de células limbocorneales dañadas. Para disminuir el riesgo que supone el trasplante de limbo en el ojo donante, se han propuesto técnicas de cultivo de células limbocorneales a partir de pequeñas biopsias limbocorneales (AU)

The cornea is a transparent tissue microscopically constituted by 5 well differentiated layers. The corneal epithelium is essential for corneal transparency and is found in a state of constant renovation throughout life on the basis of the population of limbocorneal stem cells. The localisation of these limbocorneal stem cells seems to be in the basal layers of the limbocorneal epithelium, of vital importance for maintaining the micro-environment of these limbocorneal stem cells, which depend on a variety of intrinsic and extrinsic factors. Limbic insufficiency occurs when there is a partial or total loss of these limbocorneal stem cells. These clinical features lead to a corneal clouding with a resulting loss of vision. In these cases, corneal transplant only represents a temporary replacement of the corneal epithelium; it is necessary to carry out a prior treatment involving transplant of the autologous or allogeneic limbus, which enables regeneration of the population of damaged limbocorneal cells. To reduce the risk involved in the transplant of the limbus of the donor eye, techniques of cultivation of limbocorneal cells on the basis of small limbocorneal biopsies are proposed (AU)

Localization of corneal epithelial stem cells in the developing rat.

A monoclonal antibody, 4G10.3, was developed that preferentially binds limbal basal cells in adult rat, rabbit, and human corneas. These cells were hypothesized to be the stem cells for the corneal epithelium. The antibody 4G10.3 was localized by immunofluorescence microscopy in rats 1 d and 1, 1.5, 2, 3, 4, and 6 wk of age. Until 1.5 wk, 4G10.3 bound intensely to all basal cells in the cornea and the limbus. At 2 wks, the basal cells at the central cornea abruptly changed their shape from flattened or ovoid to large and cuboidal and bound 4G10.3 with greatly reduced intensity. Increased stratification of epithelium also was seen. Cells binding 4G10.3 gradually became sequestered to the limbal area after 2 wk, concomitant with increased stratification. At 4 and 6 wk, 4G10.3 binding was identical to that in adult corneas with only limbal basal cells showing positive binding. Basal cells in the limbal epithelium did not decrease their intense binding of 4G10.3 or change their ovoid cellular shape from 1 d through adult life. These results suggest that, during development, stem or stem-like cells are localized throughout the basal layer of the corneal and limbal epithelium. As the cornea matures, these cells are sequestered in the limbus at the same time that stratification of the epithelium and shape changes occur in the basal cells.