Sai Kolli

Differentiation of Human Embryonic Stem Cells into Corneal Epithelial‐Like Cells by In Vitro Replication of the Corneal Epithelial Stem Cell Niche

Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiating into any cell type of the body. It has long been known that the adult stem cell niche is vital for the maintenance of adult stem cells. The cornea at the front of the eye is covered by a stratified epithelium that is renewed by stem cells located at its periphery in a region known as the limbus. These so‐called limbal stem cells are maintained by factors within the limbal microenvironment, including collagen IV in basement membrane and limbal fibroblasts in the stroma. Because this niche is very specific to the stem cells (rather than to the more differentiated cells) of the corneal epithelium, it was hypothesized that replication of these factors in vitro would result in hESC differentiation into corneal epithelial‐like cells. Indeed, here we show that culturing of hESC on collagen IV using medium conditioned by the limbal fibroblasts results in the loss of pluripotency and differentiation into epithelial‐like cells. Further differentiation results in the formation of terminally differentiated epithelial‐like cells not only of the cornea but also of skin. Scanning electron microscopy shows that some differences exist between hESC‐derived and adult limbal epithelial‐like cells, necessitating further investigation using in vivo animal models of limbal stem cell deficiency. Such a model of hESC differentiation is useful for understanding the early events of epithelial lineage specification and to the eventual potential application of epithelium differentiated from hESC for clinical conditions of epithelial stem cell loss.

Successful Clinical Implementation of Corneal Epithelial Stem Cell Therapy for Treatment of Unilateral Limbal Stem Cell Deficiency

The corneal epithelium is maintained by a population of stem cells known as limbal stem cells (LSCs) due to their location in the basal layer of the outer border of the cornea known as the limbus. Treatment of limbal stem cell deficiency (LSCD) has been achieved with transplantation of ex vivo expanded LSCs taken from a small biopsy of limbus. This is a relatively new technique, and as such, specific national or international guidance has yet to be established. Because of the lack of such specific guidance, our group has sought to minimize any risk to the patient by adopting certain modifications to the research methodologies in use at present. These include the replacement of all non‐human animal products from the culture system and the production of all reagents and cultures under Good Manufacturing Practice conditions. In addition, for the first time, a strictly defined uniform group of patients with total unilateral LSCD and no other significant ocular conditions has been used to allow the success or failure of treating LSCD to be attributable directly to the proposed stem cell therapy. A prospectively designed study with strict inclusion and exclusion criteria was used to enroll patients from our database of patients with unilateral LSCD. Eight eyes of eight consecutive patients with unilateral total LSCD treated with ex vivo expanded autologous LSC transplant on human amniotic membrane (HAM) with a mean follow‐up of 19 (RANGE) months were included in the study. Postoperatively, satisfactory ocular surface reconstruction with a stable corneal epithelium was obtained in all eyes (100%). At last examination, best corrected visual acuity improved in five eyes and remained unchanged in three eyes. Vision impairment and pain scores improved in all patients (p < .05). This study demonstrates that transplantation of autologous limbal epithelial stem cells cultured on HAM without the use of non‐human animal cells or products is a safe and effective method of reconstructing the corneal surface and restoring useful vision in patients with unilateral total LSCD. STEM CELLS 2010;28:597–610

LOSS OF CORNEAL EPITHELIAL STEM CELL PROPERTIES IN OUTGROWTHS FROM HUMAN LIMBAL EXPLANTS CULTURED ON INTACT AMNIOTIC MEMBRANE

BACKGROUND The corneal epithelium is renewed by stem cells located at the limbus, the so-called limbal stem cells (LSCs). Absence, damage or loss of the LSC population leads to the painful and blinding condition of LSC deficiency (LSCD). Ex vivo expansion of LSCs is an increasingly well recognized treatment modality for LSCD. One method of ex vivo expansion of LSCs involves the culture of limbal explants on amniotic membrane (AM). The purpose of this study was to analyze the outgrowths from human cadaveric limbal explants cultured on AM for properties associated with LSCs. In particular, the expression of putative stem cell markers and the colony-forming efficiency of the different zones of the outgrowth were studied. METHODS The limbal explants were expanded in the standard way used for clinical transplantation and the outgrowths were divided into three zones depending on proximity to the explant (inner, middle and outer zones). The colony-forming efficiencies (CFEs) of cells from each zone were calculated. In addition, the expression of DeltaNp63, ABCG2 (both putative positive LSC markers) and cytokeratin K3 (marker of corneal differentiation) were assessed using quantitative reverse transcription PCR (RT-PCR). Immunohistochemistry on paraffin-embedded sections was also performed to demonstrate protein localization and allow further confirmation of the quantitative RT-PCR results. RESULTS Successful cultures for both the explant outgrowths and the CFE calculations were obtained in every case. CFE showed a successive decline in zones further away from the explant (p < 0.00005). Quantitative RT-PCR revealed that the expression of the positive putative LSC markers DeltaNp63 and ABCG2 also showed a steady decrease in the zones furthest from the explant (p < 0.05 and p < 0.005, respectively). The expression of cytokeratin K3 was increased in zones furthest from the explant (p < 0.005). Immunohistochemistry on paraffin-embedded sections of intact ex vivo-expanded limbal epithelium for the putative positive marker p63 and cytokeratin K3 confirmed the findings of the quantitative RT-PCR and CFE results. CONCLUSIONS We demonstrate for the first time that outgrowths from human limbal explants, a widely used technique in ex vivo expansion of LSCs for clinical transplantation, show a steady decline in a wide range of stem cell properties with distance from the central explant. These findings support the importance of proximity of stem cells to their niche environment in maintaining their undifferentiated state. These findings suggest the need for modifications of existing techniques to ensure maximum numbers of LSCs following ex vivo expansion protocols, which will then ensure the success of subsequent engraftment.

Successful Application of Ex Vivo Expanded Human Autologous Oral Mucosal Epithelium for the Treatment of Total Bilateral Limbal Stem Cell Deficiency

Ocular surface reconstruction with ex vivo expanded limbal stem cells (LSCs) is a widely used clinical treatment for patients with limbal stem cell deficiency (LSCD). This is not applicable to patients with bilateral LSCD where there are no remaining LSCs. Cultivated oral mucosa epithelium (OME) has been used as an alternative source of autologous epithelial stem cells for ocular reconstruction in few clinical trials. However, successful generation of stratified OME epithelium has only been achieved in the presence of animal feeder cells and/or animal‐derived products in the culture media, likely to contribute to increased risk of pathogen transmission and graft rejection. In this study, we report generation of multilayered OME epithelium that shares many of the characteristics of corneal epithelium using a fully compliant good manufacturing practice, feeder‐ and animal product‐free method. Proof of concept was achieved by transplantation of autologous ex vivo expanded OME in two patients with histologically confirmed bilateral total LSCD that resulted in successful reversal of LSCD in the treated eye up to 24 months. Stem Cells 2014;32:2135–2146

A putative role for RHAMM/HMMR as a negative marker of stem cell-containing population of human limbal epithelial cells

The corneal epithelium is maintained by stem cells located at the periphery of the cornea in a region known as the limbus. Depletion of limbal stem cells (LSCs) results in limbal stem cell deficiency. Treatments for this disease are based on limbal replacement or transplantation of ex vivo expanded LSCs. It is, therefore, crucial to identify cell surface markers for LSCs that can be used for their enrichment and characterization. Aldehyde dehydrogenases (ALDHs) are enzymes which protect cells from the toxic effects of peroxidic aldehydes. In this manuscript, we show for the first time that ALDH1 is absent from the basal cells of the limbal and corneal epithelium. We separated limbal epithelial cells on the basis of ALDH activity and showed that ALDHdim cells expressed significantly higher levels of ΔNp63 and ABCG2 as well as having a greater colony forming efficiency (CFE) when compared to ALDHbright cells. Large scale transcriptional analysis of these two populations led to identification of a new cell surface marker, RHAMM/HMMR, which is located in all layers of corneal epithelium and in the suprabasal layers of the limbal epithelium but is completely absent from the basal layer of the limbus. Our studies indicate that absence of RHAMM/HMMR expression is correlated with properties associated with LSCs. RHAMM/HMMR‐ limbal epithelial cells are smaller in size, express negligible CK3, have higher levels of ΔNp63 and have a higher CFE compared to RHAMM/HMMR+ cells. Taken together these results suggest a putative role for RHAMM/ HMMR as a negative marker of stem cell containing limbal epithelial cells. Cell selection based on Hoechst exclusion and lack of cell surface RHAMM/HMMR expression resulted in increased colony forming efficiency compared to negative selection using RHAMM/HMMR alone or positive selection using Hoechst on its own. Combination of these two cell selection methods presents a novel method for LSC enrichment and characterization.

Corneal Epithelial Stem Cells and Their Therapeutic Application

The cornea is the clear window at the front of the eye and its clarity is vital for the transmission of light to the retina at the back of the eye for visual perception. The surface of the cornea is made up of an epithelium, which is continuous with that of the surrounding conjunctiva. The transition between the corneal and conjunctival epithelia is formed by the limbal epithelium. The limbal epithelium has two particular important functions. First, it harbours the corneal epithelial stem cells (CESCs), also known as limbal stem cells (LSCs). These stem cells (SCs) provide a reservoir for corneal epithelial cells, which are needed to replace those lost continuously from the corneal surface. Second, it acts as a barrier to prevent the phenotypically and functionally different conjunctival epithelium from encroaching onto the corneal surface, which would impair the transparency of the cornea and lead to visual loss.