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Abstract

The ocular surface is the outermost part of the visual system that faces many extrinsic or intrinsic threats, such as chemical burn, infectious pathogens, thermal injury, Stevens–Johnson syndrome, ocular pemphegoid, and other autoimmune diseases. The cornea plays an important role in conducting light into the eyes and protecting intraocular structures. Several ocular surface diseases will lead to the neovascularization or conjunctivalization of corneal epithelium, leaving opacified optical media. It is believed that some corneal limbal cells may present stem cell-like properties and are capable of regenerating corneal epithelium. Therefore, cultivation of limbal cells and reconstruction of the ocular surface with these limbal cell grafts have attracted tremendous interest in the past few years. Currently, stem cells are found to potentiate regenerative medicine by their capability of differentiation into multiple lineage cells. Among these, the most common cell sources for clinical use are embryonic, adult, and induced stem cells. Different stem cells have varied specific advantages and limitations for in vivo and in vitro expansion. Other than ocular surface diseases, culture and transplantation of corneal endothelial cells is another major issue for corneal decompensation and awaits further studies to find out comprehensive solutions dealing with nonregenerative corneal endothelium. Recently, studies of in vitro endothelium culture and ρ-associated kinase (ROCK) inhibitor have gained encouraging results. Some clinical trials have already been finished and achieved remarkable vision recovery. Finally, nanotechnology has shown great improvement in ocular drug delivery systems during the past two decades. Strategies to reconstruct the ocular surface could combine with nanoparticles to facilitate wound healing, drug delivery, and even neovascularization inhibition. In this review article, we summarized the major advances of corneal limbal stem cells, limbal stem cell deficiency, corneal endothelial cell culture/transplantation, and application of nanotechnology on ocular surface reconstruction. We also illustrated potential applications of current knowledge for the future treatment of ocular surface diseases.

Keywords

Corneal neovascularization Stem cells Nanomedicine Regeneration medicine Corneal endothelial cell transplant

Introduction

The cornea is the guardian of the eye with avascularity and transparency that are both vital for normal vision. The epithelium is the outermost part of the cornea, followed by Bowman's layer, stroma, Descemet's membrane, and endothelium. The corneal epithelium is continuous with the conjunctival epithelium at the corneolimbal area and is responsible for maintaining the smoothness and integrity of the ocular anterior surface. The shed corneal epithelial cells are regenerated from stem cells at the limbus; therefore, severe limbal stem cell (LSC) deficiency may cause conjunctival invasion due to loss of cellular barrier, which in turn leads to corneal opacification that results from the vascularized entity of conjunctiva (59,92). The thickest part of the cornea is the stroma, and its transparency relies on regularly arranged collagen fibers. The endothelial cells are the innermost part of the cornea, responsible for fluid and solute transport between the corneal stroma and aqueous humor. Unfortunately, the cells of the endothelium cannot regenerate. If the density of endothelial cells becomes too sparse, the stroma will thicken and lose its transparency, which may provoke corneal edema with the opacified sequelae and thus impair the visual image formed.

Corneal Stem Cells, Limbal Stem Cell Deficiency, and Stem Cell Therapies

Limbal Stem Cells and Associated Genetic Expression

The limbus, located at the 2-mm-wide area between the cornea and the bulbar conjunctiva, is considered to be the niche of corneal epithelial stem cells (14,31,75). The basal epithelial cells near the limbus are not homogeneous, but mixed with a diverse population of stem cells, transient amplifying cells (TACs), and terminally differentiated cells (93). The TACs are first generated by stem cells and then migrate to the central cornea and proliferate rapidly afterward.

Majo et al. transplanted the cornea or limbus from β-gal-ROSA26 mice into athymic mice and found that oligopotent stem cells are distributed throughout the mammalian ocular surface (49). They noticed that the limbus has no contribution to the steady-state renewal of the corneal epithelium but becomes instrumental when the cornea was severely wounded. Their results challenged the idea that the limbus is the sole niche for corneal stem cells. They further postulated that stem cells move from the conjunctival and corneal side of the limbus and accumulate in the limbus (49).

ATP-binding cassette subfamily G member 2 (ABCG2), p63, integrin a 6, K19, N-cadherin, nerve growth factor (NGF) receptors (TrkA), octamer-binding transcription factor 4 (OCT4), αvβ5-integrin, and CXCL10/IP-10 have all been proposed as candidate markers to identify limbal stem cells (6,18,27,28,65,67,68,76,89,98,111). The p63 gene, also called TP63, is located in chromosome 3q27–29. Transcription from different promoters of the TP63 gene can generate two different premessenger RNAs: TAp63 and δNp63. Alternative splicing of each transcript can produce α, β, and γ isoforms (100). All of the three isoforms are present in corneal keratinocytes in the limbus; however, δNp63α is the most abundant one (6,18,67). Protein p63 maintains the proliferative potential of LSCs. Rama et al. found in cultivated limbal epithelial transplantation (CLET) that the successful generation of normal epithelium on the patients’ stroma was associated with the percentage of p63-bright stem cells in culture (73). If these cells are more than 3% of the total number of clonogenic cells, the transplantation was successful in 78% of patients. On the contrary, the transplantation was only successful in 11% of patients when such cells were 3% or less of the total number of cells. Otherwise, a side population (SP) phenotype, based on the ability to efflux Hoechst 33342 dye, has been shown in stem cells (22). The ATP-binding cassette transporter Bcrp1/ABCG2 is reported to contribute to the SP phenotype in LSCs (98), indicating that ABCG2-positive limbal epithelial cells are putative corneal epithelial stem cells. Hayashi and his coworkers found a minor subpopulation of limbal epithelial cells presenting a higher clonogenic capacity and expression of stem cell markers with a lower expression of differentiation markers than the matured corneal epithelial cells. This subpopulation of limbal epithelial cells showing high integrin α 6 without CD71 was localized in the basal part of the limbal epithelium where corneal LSCs are believed to exist (27). N-cadherin is a member of the classic cadherin family and mediates cell-to-cell adhesion (87,99). N-cadherin was found to be expressed by putative stem/progenitor cells in the human limbal epithelial stem cell niche to prevent cell proliferation. Therefore, N-cadherin is a critical adhesion molecule between corneal epithelial stem/progenitor cells and their surrounding niche cells (28). NGF is a member of the neurotrophin family (9,50), which has many biological effects on stem cells outside the nervous system (16). Through tyrosine kinase (Trk) receptors (TrkA), NGF can stimulate corneal epithelial proliferation. Owing to preferential localization of TrkA to limbal basal epithelial cells, it is considered as a potential marker of corneal LSCs (41,84,90). Transcription factor OCT4 is crucial for self-renewal and maintenance of embryonic stem cells. Zhou et al. found the OCT4 gene is expressed mainly in the basal layer of the human corneal epithelium, especially in the limbal area (111). These OCT4-positive corneal epithelial basal cells have the potent capacity of proliferation. They suspected that the transcription factor could control the phenotype of different layers of corneal epithelium. Vitronectin (VN) is mainly expressed in the human limbus but not the corneal basement membrane. It can enhance colony efficiency of limbal epithelial cells (19). Ordonez et al. postulated that VN receptors (integrins αvβ3/5) can be used to identify and isolate limbal epithelial stem cells (LESCs) (65). They led a study and found that the expression of integrin αvβ5 is restricted to the limbus, and αvβ5⁺ limbal epithelial cells have the same phenotypic and functional properties as LESCs. They thus concluded that integrin αvβ5 is a candidate LESC marker. They also confirmed that αvβ5⁺ basal limbal epithelial cells coexpress CXCL10/IP-10.

Limbal Stem Cell Deficiency (LSCD) and Various Associated Treatments

LSCs are important for maintaining the corneal epithelium, which is preferentially vascularized under the condition of LSCD. There are several conditions that lead to LSCD: congenital aplasia of stem cells (such as aniridia), disease with extrinsic causes (such as chemical or thermal injury), ionizing radiation, repeated surgical interventions, extensive microbial infection, long-term use of contact lenses, and disease originated from intrinsic causes (such as Stevens– Johnson syndrome) (35). The characteristics of LSCD are conjunctivalization and neovascularization of the cornea, irregularity of the corneal surface (calcification, ulceration, melting, and perforation of cornea), recurrent/ persisted epithelium defect, and subepithelial scarring (71,80).

Improving a healthy ocular surface is the first step in the management of LSCD. In partial or total LSCD, a better environment of ocular surface helps the remaining LSCs or the transplanted limbal graft survive. Frequent administration of preservative-free artificial tears, topical cyclosporine, steroids, and punctal occlusion may be used to treat the combined dry eye syndrome and ocular surface inflammation. In some cases, scleral lenses can help to improve vision, reduce ocular pain, and promote healing of persisted epithelial defects that are refractory to medical treatments (77,83). In patients with refractory partial LSCD, surgical interventions such as repeated debridement and amniotic membrane transplantation should be considered (4). The amniotic membrane (AM) is able to promote corneal reepithelialization by preserving and maintaining the epithelial progenitor cells and also reduce angiogenesis and inflammation (53,94). Autologous limbal transplantation from the healthier eye can be performed in patients with unilateral LSCD. For patients with unilateral or bilateral deficiencies, allogeneic limbal grafts can be performed to reconstruct the corneal surface.

Owing to the shortage of donor limbal grafts, many scientists tried to cultivate LSCs for transplantation (51,73,91). Tsai and his colleagues tried to perform a limbal biopsy of the epithelial layer and the corneal stroma 1 by 2 mm on the contralateral healthy eye in six patients with hormonal epithelial medium and 5% fetal calf serum on the basement membrane side of the AM for 2 to 3 weeks to expand the limbal epithelial cells to an area 2 to 3 cm in diameter. The transplanted limbal epithelial cells plus AM on sectorial or circular lesion sites (90° to 360°) of the LSCD patients’ damaged corneal surface successfully restored the corneal epithelium, and the clarity of the cornea was also improved (91). In clinical trials, the autologous cultured limbal epithelial cell transplantation can restore the corneal surface and improve the visual acuity in patients with LSCD (66,104). Improvement of visual acuity was noted in 73% of patients during a mean follow-up period of 28.5 months in Pauklin et al.'s study and in 83% patients with a 15-month study period in Tsai's report. Rama and his colleagues successfully used fibrin to culture autologous LSCs to treat chemical and thermal burn-related LSCD without any severe side effects (73). Their results proved the efficacy of cultivated limbal epithelial cell transplantation and avoided the risk of iatrogenic LSCD in the healthy eyes.

In patients with bilateral LSCD, a sufficient amount of limbal cells is difficult to obtain for tissue expansion. Instead, cultured oral mucosal epithelial cells for transplantation on the corneal surface are being considered and are gaining much attention (58,60). During the engineering of oral mucosal epithelial cell sheets for ocular surface reconstruction, the retrieved mucosal cells were isolated into cell suspensions and were cultivated with mitomycin C-treated 3T3 feeder cells on AM or a specially designed plate. Several characteristics of ex vivo cultured oral mucosal cell sheets are similar to the human corneal epithelium, including stratified three to five layers of cells, round and small basal cells, flat middle and superficial cells, apical microvilli, and good transparency. Typical markers of the human corneal epithelium, such as keratin 3, p63, and β-integrin, were also identified in cultured oral epithelial cell sheets. In a rabbit model of LSCD, autologous transplantation of rabbit oral mucosal epithelial cells to destructed ocular surfaces resulted in smooth reconstructed surface and recovered transparency in this short-term study (57). Nishida et al. further successfully transplanted cultivated oral mucosal epithelial cells on the damaged ocular surface of patients with Stevens–Johnson syndrome and ocular cicatricial pemphigoid and achieved complete reepithelialization of the corneal surfaces in all four treated eyes without any complications (60). In their study, the autologous oral mucosal epithelial cells were cultivated on temperature-responsive inserts with a doughnut-shaped supporter, designed for separation of 3T3 feeder cells. The epithelial cell sheets were transplanted onto the denuded ocular surface without suture. The procedures of cultivating LSCs and oral mucosal epithelial cells for transplantation are demonstrated in Figure 1.

Figure 1.

Cultivated limbal stem cells and oral mucosal epithelial cells. (A) Epithelial cells could be cultivated from contralateral healthy limbus. Small (1 by 2 mm) specimens of healthy limbal epithelium and superficial stroma were harvested, treated, and cultured on basement membrane side of amniotic membrane for 2 to 3 weeks. When the epithelial cells grew to 2 to 3 cm in diameter, the basement membrane with epithelial cell sheets could be transplanted on the cornea surface. (B) Epithelial cells could be cultivated from oral mucosa with a special culture system. In this system, the feeder 3T3 cells were pretreated with mitomycin C (MMC), which deprived their proliferative activities and were then seeded onto tissue culture wells. Small (3 by 3 mm) specimens of oral mucosal tissue were harvested and treated with trypsin and EDTA. The isolated epithelial cells were then seeded on temperature-responsive cell culture inserts and cultured in these tissue culture wells of feeder 3T3 cells. Epithelial cells would grow as multilayered cell sheets and then be separated from the culture systems by reducing temperature. These cell sheets could be transplanted on the cornea directly.

Stem Cell Therapies for Ocular Surface Diseases

Embryonic Stem Cells (ESCs), Adult Stem Cells, and Induced Pluripotent Stem Cells (iPSCs)

ESCs are pluripotent stem cells derived from the inner cell mass of a blastocyst that have the ability to differentiate into all three germ layers and their progenitors. With this pluripotent ability, ESCs are good potential cell sources for regenerative therapies. Since the protocol of differentiating human ESCs (hESCs) into corneal epithelium-like cells is still being debated, studies have been trying to elucidate the best niche for hESCs to form corneal epithelium. Collagen IV-coated culture plates with conditioned medium from limbal fibroblast promotes hESC fate to become corneal epithelium-like cells. Both early epithelial progenitor cells with peak p63 expression and more differentiated corneal-like epithelial cells with prominent cytokeratin (CK) 3/12 and CK12, markers of mature corneal epithelial cells, were observed in the study (3). The expression of stage-specific embryonic antigen 4 (SSEA-4) is observed both in hESC-derived epithelium-like cells and ex vivo cultured limbal stromal cells. However, its association with specific differentiation of corneal cells still needs to be identified (3,44). Some adult stem cells also show ESC-like properties and may be used as alternative cells for regenerative medicine. For example, Yang et al. isolated epidermal adult stem cells expressing ESC immunological markers from the ear skin of a goat and then induced the stem cells to form embryoid bodies (101). Although ESCs show the ability to differentiate into multiple lineage cells, such as corneal progenitor cells and their downstream cells, there are always ethical and immunological issues surrounding ESC therapies. Fortunately, this limitation is now bypassed by the introduction of induced pluripotent stem cells (iPSCs). iPSCs are generated from somatic cells by the induction of transcriptional factors—OCT4, sex-determining region y-box 2 (Sox2), c-Myc, and Kruppel-like factor 4 (Klf4) show similar characters of ESCs and can potentiate many patient-specific therapies with reduced risk of immune reactions.

Differentiation of Pluripotent Stem Cells

ESCs and iPSCs are both candidate cell sources for regenerative medicine. Direct transplantation of ESCs to damaged or dysfunctional sites is a probable method to treat degenerative diseases. However, the formation of teratomas and immune reactions are possible risks that are cause for concern. For instance, positive SSEA-4 (a differentiation marker) was noted in some hESC-derived corneal epithelial-like cells in Ahmad et al.'s study, implying possible future differentiation or oncogenesis during ESC cell therapy (3). Meanwhile, life-long immunosuppression is required in patients receiving cell therapy by hESCs due to their immunogenic properties. Compared to ESCs, mature somatic cells or their progenitor cells differentiated from iPSCs might be safer for clinical use. Based on the concept of producing iPSCs, there should be numerous candidate sources of somatic cells for reprogramming, such as skin fibroblasts, the most commonly utilized cells. However, different lineages of somatic cells for iPSCs showed various propensities for further corneal epithelial differentiation in Nishida and colleagues’ study, demonstrating that cell sources from corneal limbal epithelial cells are better than those from skin fibroblast to produce epithelial-like cells (26). This finding has been attributed to the epigenetic modification on the genes dealing with iPSC differentiation. In order to treat LSCD or other ocular surface diseases safely, ESCs or iPSCs should be differentiated into corneal epithelium or LSCs first. The functions and stemness of LSCs are highly associated with the microenvironments, including the extracellular matrix and the paracrine of nearby fibroblasts. This provided us with a way to guide the epithelial differentiation of pluripotent stem cells by manipulating the microenvironments to be similar to the corneal LSC niche. Corneal LSCs are normally situated in the limbal stroma, mixed with complex limbal niche cells (LNCs) and extracellular matrix, mainly type IV collagen. In vitro coculture of limbal epithelial progenitor cells (LEPCs) with LNCs showed maintained clonal growth of corneal progenitor cells and less epithelial differentiation. The underlying mechanism may be ascribed to balanced BMP/Wnt signals, secreted by LEPCs and LNCs (23).

The most popular method for in vitro epithelial differentiation is to use collagen IV-coated plates and conditioned medium from limbal fibroblasts (2,3,97). The conditioned medium of fibroblast contains the keratinocyte growth factor (KGF), a stimulator for LSCs (82). With the assistance of collagen IV-coated plates and limbal fibroblast-conditioned medium or DMEM/F12-conditioned media (DF-CM), corneal epithelial progenitors expressing E-cadherin, CD44, p63α, and ABCG2 can be generated (29,40,112). These corneal epithelial progenitors can form a monolayer (29) or multilayers (40,112) of epithelial-like cells that adhere on corneal stroma in animal models. The epithelial progenitor cells can be directly transplanted (29,40) or seeded on an acellular porcine corneal matrix (APCM) as stratified epithelial cell sheets first and utilized later for transplantation (112). Ueno et al. tried transfecting the Pax6 gene to differentiate mouse ESCs into corneal epithelium-like cells (96). Pax6 is an essential transcriptional factor in the embryonic development of the cornea and anterior segment of the eye (17). When these Pax6-transfected cells were transplanted onto damaged corneas, they could adapt to the injured cornea and remained alive on it.

Other researchers directly transplanted ESCs or spontaneously differentiated ESCs and let these cells differentiate naturally in an in vivo microenvironment. Hanson et al. simply cultured human ESCs with differentiation medium and allowed them to spontaneously differentiate (24). They later transplanted these differentiated human ESCs on wounded human corneal buttons in vitro and successfully generated one to four layers of epithelial cells expressing Pax6 on day 3 and Pax6 with CK3 on day 6. Zhang et al. even developed a quick method to form scaffold-free embryonic stem cell sheets (SESCSs) for transplantation (107,108). They suspended ESCs in a glycerin medium and constructed cell layers on AM. When SESCSs were transplanted in a rabbit with LSCD, they repaired corneal damage and further differentiated into three groups of cells: corneal LSCs, corneal transient amplifying cells, and terminally differentiated cells. These results demonstrated that the in vivo microenvironment is conducive for the differentiation of ESCs into epithelial cells.

Mouse (102) and human (26) iPSCs are also differentiated into corneal epithelium successfully with the stromal cell-derived inducing activity (SDIA) method. Bone morphogenetic factor 4 (BMP4), participating in surface ectodermal differentiation and suppression of neural differentiation, belongs to the transforming growth factor-β (TGF-β) superfamily of proteins. Although BMP4 may generally potentiate ectodermal differentiation, early suppression of iPSC differentiation to corneal epithelium was observed in Hayashi et al.'s study (26) with decreased expression of Pax6 and K12, but δNp63 was left unaffected. On the contrary, Shalom-Feuerstein and his coworkers found that BMP4, corneal fibroblast-derived conditioned medium, and collagen IV could robustly differentiate iPSCs into epithelial cells (79). They also found that the presence of miR-450b-5p could repress Pax6 and trigger epidermal differentiation, while its absence could preserve the activity of Pax6 for corneal epithelial differentiation. The discrepant results about the function of BMP4 in corneal epithelial differentiation may result from the period of exposure, original cell types to be differentiated, and different concentrations of BMP4. More studies are required to identify the role of BMP4. Type IV collagen is the main component of the basement membrane in ocular tissues, such as corneal epithelium and retinal pigment epithelium. Thus, coating collagen IV on culture dishes helps differentiation and arrangement of corneal epithelium. Yu et al. found that coculture of mouse iPSCs with corneal limbal stroma in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and NGF could also help iPSCs differentiate into corneal epithelial-like cells (103). The procedures of differentiating pluripotent stem cells into corneal epithelial cells are demonstrated in Figure 2.

Figure 2.

Differentiation of pluripotent stem cells (PSCs) into corneal epithelial cells. (A) In this method, pluripotent stem cells (PSCs) were cultured on collagen IV-coated plates with limbal fibroblasts conditioned medium. Collagen IV is an important component in basement membrane. Limbal fibroblasts conditioned medium contains keratinocyte growth factor (KGF) and many other factors. With the environment that mimicked the niche of corneal epithelial stem cell, PSCs could then differentiate into epithelium-like cells. Some researchers even added bone morphogenetic factor 4 (BMP4) as an enhancer for ectodermal differentiation. (B) Stromal cell-derived inducing activity (SDIA) method was another strategy for the epithelial differentiation of PSCs. In this method, PSCs were isolated and cultured with the feeder fibroblasts, which were pretreated with mitomycin C. Feeder fibroblasts could provide some factors to guide the differentiation of PSCs into epithelial-like cells. Some researchers also enhanced the effect of epithelial differentiation by transfecting Pax6 gene into PSCs to provide these essential transcriptional factors or by adding BMP4 into the media. (C) The third method for epithelial differentiation of PSCs was mediated by in vivo microenvironment. When PSCs were cocultured with limbal stromal cells in the media containing some important factors, such as bFGF, EGF, or NGF, spontaneous differentiation into epithelium-like cells could be observed.

Other Applications of Pluripotent Stem Cells

Different methods have also been investigated to repair corneal damage by coculturing corneal epithelial cells with ESCs or their condition medium so as to increase the stemness and the healing functions of ESCs. Zhou et al. showed that corneal epithelial cells cocultured with ESCs increased the expression of stem cell markers ABCG2 and p63 and decreased the differentiation marker, keratin 3 (110). These cells showed elevated cell functions through the activation of the integrin, β1-FAK-PI3K/Akt, signaling pathway and could regenerate a functional stratified corneal epithelium. Zhan et al. modulated rabbit corneal epithelial cells by incubating these cells with murine ESC extract (106). In their study, transiently reprogrammed epithelial cells with positive alkaline phosphatase (AKP) staining and upregulated OCT4/SSEA-1 were observed. Nonetheless, the reprogrammed effect could not last for a long time; meanwhile, ESC markers were also down-regulated gradually. On the other hand, ESC conditioned media-mediated enhancement of stemness and cellular proliferation were also observed in human LSC culture and rabbit epithelial cells (45,46).

Some researchers proved that iPSCs could not merely be used for tissue regeneration, but might also be utilized to help the healing of the cornea in other ways. Chien et al. demonstrated that iPSCs derived from human corneal keratocytes with the delivery system of amphiphatic carboxymethyl-hexanoyl chitosan (CHC) hydrogel could enhance corneal reconstruction by downregulating oxidative stress and recruiting endogenous epithelial cells in a corneal alkaline damage model (10). Shalom-Feuerstein et al. investigated potential therapeutic agents for ectodermal dysplasia-induced LSCD (p63 gene mutation) with a patient-specific iPS cellular model (78). They found iPSCs reprogrammed from patients’ fibroblasts could differentiate into K18-positive cells but fail to further differentiate into K14-positive cells (epidermis/limbus) or K3/K12-positive cells (corneal epithelium). APR-246 [PRIMA-1(MET)], a small compound that can restore functionality of mutant p53 in human tumor cells, could reinstate the p63-related signaling pathway in this iPS cellular model. They concluded that their findings paved a way for future therapies of ectodermal dysplasia-related patients.

Corneal Stromal Stem Cells

Although LSCs are applied exclusively to the corneal epithelial progenitors, both corneal epithelial and stromal stem cells existed in the corneal stem cell niche (25). The corneal stroma is composed of collagen fibers and keratocytes, which exhibit a dendritic morphology (70). Branch et al. demonstrated that cultured stromal cells of the limbus and peripheral cornea (PLCSCs) can produce a mesenchymal stem cell (MSC) population (7). These MSCs may provide a supportive niche for epithelial stem cells (43). Hashmani et al. found that PLCSCs could be divided into CD34⁺CD105⁺, CD34^–CD105⁺, and CD34^–CD105^– subpopulations. CD34⁺CD105⁺ cells were the most stemlike and have a capacity for mesenchymal and epithelial differentiation (25). This discovery challenges current perceptions of the role of the keratocyte. In the future, corneal stromal stem cells may be an alternate cell source for the treatment of LSCD.

Corneal Endothelial Transplant and Cell Therapy

Corneal Endothelial Diseases and Associated Modern Keratoplasty

Since the corneal endothelial cells can only enlarge and migrate themselves to compensate for the decreasing cell density rather than regenerate themselves when damage happens, severe corneal endothelial diseases tend to cause corneal edema, decompensation, and loss of transparency. Corneal endothelial keratoplasty is a developing trend in solving corneal endothelial diseases that are not responsive to medical treatment, such as Fuch's endothelial dystrophy and bullous keratopathy. The two major procedures of endothelial keratoplasty are Descemet membrane endothelial keratoplasty (DMEK) and Descemet's stripping automated endothelial keratoplasty (DSAEK), both of which are aiming at replacement of the diseased corneal endothelium with a thin donor endothelial cell layer and retaining healthy anterior lamella of the cornea in situ. Compared to traditional penetrating keratoplasty (PK), endothelial keratoplasty presents a preferable surgical outcome, less graft rejection, and minor residual astigmatism. However, shortage of donor tissue and high technique dependence also limit its availability. Hence, in vitro human corneal endothelial cell (HCEC) cultures are deeply studied now for endothelial transplant, though there is no consensus about culture protocol of HCECs so far.

In Vitro Expansion of Corneal Endothelial Cells

Pericellular matrix from human decidua-derived mesenchymal cells (PCM-DM) has been used by Numata et al. as a substrate on which the monkey corneal endothelial cells are cultured. Improved cell adhesion, cell density, and characteristics of corneal endothelial cells are observed in the cells with the PCM-DM substrate (61). According to Numata et al.'s in vivo study, the enhancement of CEC culture on PCM-DM may be associated with integrin, which increases CEC adhesion to substrate and is regarded as beneficial for environmental sensing of CECs. Moreover, components of PCM-DM, such as fibronectin and collagen IV, may also contribute to the organization of CECs. The advantages of using PCM-DM are its easy access and xeno-free identity for CEC culture. Collagen sheets and AM are other choices of substrate on which HCECs can be cultivated and then prepared for transplantation (32,55). Besides improving cultivating material, other researchers try to use human amniotic fluid or conditioned medium from human bone marrow MSCs to optimize in vitro HCEC expansion. Rapid cell proliferation and highly expressed characteristic surface proteins ZO-1 and Na⁺/K⁺ ATPase of endothelial cells have been observed (20,56). Furthermore, inhibition of TGF-β signaling during in vitro culture enables HCECs to grow while maintaining their normal physiological function, such as barrier and pump function, without undergoing fibroblastic change (62). The procedures of expanding corneal endothelial cells are demonstrated in Figure 3A.

Figure 3.

Endothelial cell expansion and transplant. (A) In vitro expansion of corneal endothelial cells could be done by the following steps. First, strip endothelial cells and Descemet's membrane from donor cornea and digest with collagenase A. Isolated endothelial cells are then seeded on pericellular matrix of mesenchymal stem cells (MSCs) or endothelial cells or on collagen IV-coated plate. Primary cultured endothelial cells are harvested and seeded on collagen sheets or amniotic membranes with or without the assistant of MSC conditioned medium. The expanded corneal endothelial cell sheets on the membranes are now ready for transplant. (B) Endothelial cell transplant should be started from the removal of dysfunctional endothelial cells and Descemet's membrane from the diseased cornea. Then cultivated endothelial cell sheets are inserted into anterior chambers carefully followed by the injection of large air bubble as tamponade. There is an alternative method for endothelium transplant by the injection of endothelial cell directly with the assistance of a ρ-associated protein kinase (ROCK) inhibitor, such as Y-27632. (C) ROCK is a multifunction kinase, which is involved in cell adhesion, migration, stress fiber formation, or even the regulation of cell proliferation and apoptosis. This schema shows the role of ROCK in the adhesion of endothelial cells with Descemet's membrane during transplant. ROCK could inhibit myosin light chain (MLC) phosphotase and active LIM kinase (LIMK). These effects enhance actomyosin contraction and reduced cell adhesion. ROCK inhibitor blocks the function of ROCK and could enhance cell adhesion. This is the reason why ROCK inhibitor could be useful for the transplantation of endothelial cells.

Endothelial Cell Transplant

Two brand new surgical procedures using cultivated corneal endothelial cells for advanced dysfunctional corneal endothelium have been developed. One is transplantation of in vitro cultivated corneal endothelial cell sheets grown on type I collagen carriers; the other is intracameral cell-injection therapy (38). To improve the success rate of endothelial cell transplantation and reduce substrates used in the procedure, use of a ρ-associated kinase (ROCK) inhibitor has been studied in animal models and has demonstrated benefits in promoting endothelial cell adhesion, proliferation, and in inhibiting apoptosis (63,64). Similar to the function of PCM-DM mentioned previously, the ROCK inhibitor reduces the negative regulation of ROCK on CEC adhesion to Descemet's membrane (basement membrane) by increasing expression of ZO-1, Na⁺/ K⁺-ATPase, and vinculin, a membrane-cytoskeletal protein connecting to intracellular actin. After treatment with ROCK inhibitor, improvement of cell arrangement and corneal thickness were found in a primate model (63). In rabbit and primate corneal endothelial dysfunction models, the cornea restores transparency within 2 days and 1 week, respectively, after injection of cultured corneal endothelial cells with ROCK inhibitor. This therapeutic effect with the appearance of an endothelial monolayer lasts for at least 2 weeks and 3 months in rabbit and monkey models, respectively. No abnormal intraocular cell adhesion or elevated intraocular pressure was observed in these studies. Treatment of in vitro cultured corneal endothelial cells and ex vivo corneal button with ROCK inhibitor demonstrated no toxicity and influence on cell viability (69). The procedures of corneal endothelial cell transplantation are demonstrated in Figure 3B. Application of the ROCK inhibitor as eye drops to treat patients with corneal endothelial dysfunction has already been performed in clinical trials. Quick response to treatment within 2 weeks with persistent corneal clarity for the following 2 years has been documented in a case report (39). Application of the ROCK inhibitor to patients with varying degrees of endothelial dysfunction in another clinical trial showed more obvious effects and corneal thickness recoveries in patients with focal/central corneal edema, but not in those who suffered from diffused corneal decompensation after a 6-month follow-up study (63). This result implies that the benefits of using eyedrops of ROCK inhibitor to treat corneal endothelial dysfunction may be limited to mild patients with only central corneal edema, while patients with severe endothelial dysfunction may need lamellar or cell transplant with/without ROCK inhibitor to further improve the success rate. Although the convincing therapeutic responses are found in animal and human clinical trials, long-term follow-up and large-scale clinical trials are necessary to clarify the life-long effect and possible complications.

Potential Corneal Endothelial and Trabecular Meshwork Stem Cells

Stem cells and progenitor cells in ocular tissues are often identified in the transition area near mature and well-differentiated cells, replenishing damaged or sloughed tissues. Although stem cells of corneal epithelial progenitors are identified in the limbal area, whether corneal endothelial stem cells exist is still under debate. Trabecular meshwork (TM) and CECs share the same embryonic origin from neural crest and are continuous with each other. Thereafter, potential stem cells or progenitor cells are supposed to be located in the area between the peripheral endothelium and anterior nonfil-tering TM, called Schwalbe's ring region, according to clinical observation and in vivo animal studies.

The Schwalbe's line cells, which act as modulators in the anterior chamber, have several unique properties, such as immunoreaction to neuron-specific enolase (NSE), production of hyaluronan, and reaction to argon laser trabeculoplasty (ALT) (1,74,85). Since some CEC regeneration was noted in patients with graft detachment after receiving endothelial keratoplasty and in animal models of endothelial loss, a transition zone has been proposed as a stem cell niche for endothelial regeneration.

Laboratory evidence also supports the concept that stem cell-like cells occur in the transition area. Stem cell markers, including telomerase activity, nestin, alkaline phosphatase, OCT3/4, Wnt 1, Pax6, and Sox2, were observed in the peripheral cornea of animals with wounded endothelium (52). However, it is impractical now to find a specific cell signature to isolate endothelium or trabeculum-specific stem cells from the transition area. Thus, sphere culture protocol is suggested to identify progenitor cells, which show the ability of self-renewal and potential to differentiate into neuronal lineages (13). It could be beneficial to harvest possible progenitor cells of corneal endothelium to directly restore malfunctioned endothelial cells or to be used in producing iPSCs for further cell expansion and differentiation.

Nanotechnology in Corneal Disease

Nanodrugs in Corneal Diseases

Nanotechnology involves the creation and use of materials/devices at the size scale of intracellular structures and molecules and involves systems and constructs in the order of lt;100 nm (105). The major objectives of nanomedicine are monitoring, controlling, and curing diseases at a molecular level. These nanoscaled substances are of unique functions at the cellular, atomic, and molecular levels. In addition, nanotechnology provides new drug delivery treatments in a more efficient manner: delivering of pharmaceutical active ingredients at a single cellular level. With tremendous advances in nanotechnology in the past two decades, scientists and ophthalmologists have been focusing on developing new nanoparticulated formulations or drug delivery platforms for ocular medications and transplantations (i.e., treatment against oxidative stress, controlling intraocular pressure, treating neovascularization, and curing retinal degenerative diseases with gene therapy). Generally, ocular therapeutics have been administered to the eyes as an aqueous solution. However, nearly 90% of the dose applied topically from eyedrops is lost due to precorneal losses (lacrimation and drainage), which results in poor ocular availability (34). Therefore, an applicable delivery system that can prolong the contact time of the drug on the ocular surface and help drug molecules penetrate into the ocular tissue is desired. To achieve this goal, nanoparticle-based, controlled (or sustained) delivery systems for ophthalmic drugs have been considered beneficial in the past few years.

Corneal Neovascularization

The cornea can maintain its avascularity and clarity without blood and lymphatic vessels to supply oxygen and nutrients, which are mainly supported by the tear and aqueous. However, corneal neovascularization (NV), also named corneal angiogenesis, does happen and causes opacification of the ocular surface under some pathological conditions, including contact lens wearing, infectious keratitis, Stevens–Johnson syndrome, corneal graft rejection, LSCD, and chemical burn. The mechanisms that may be involved in NV include 1) vasculogenesis, the formation of new blood vessels from bone marrow-derived angioblasts (mostly during embryogenesis) and 2) angiogenesis, the formation of new vessels from preexisting vascular structures (21). These complicated situations result in corneal hypoxia, infection, inflammation, and poor limbal barrier function, respectively (15,54,95). Thus, people who wear contact lenses, patients that have had corneal transplants, and those who are suffering from infectious ocular diseases are the most affected populations of NV. An inclination from antiangiogenic toward angiogenic status finally results in neovascularization (8) that is thought to be a part of the wound healing process to bring nutrients and immune cells but results in scar formation and lipid deposition (42,86). The angiogenic factors detected in the cornea were fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), pla centa growth factor (PGF), transforming growth factor-α (TGF-α), TGF-β, insulinlike growth factor (IGF), leptin, integrins (avb3), platelet-derived growth factor (PDGF), matrix metalloproteinases (MMPs), angiogenin, hepatocyte growth factor-scatter factor (HGF-SF), tumor necrosis factor-α (TNF-α), connective tissue growth factor (CTGF), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) (8). On the other side, the antiangiogenetic factors found in the cornea were endostatin, angiostatin, prolactin, MMPs, tissue inhibitor of matrix metalloproteinase (TIMPs), thrombospondin, arresten, canstatin, tumstatin, pigment epithelium-derived factor (PEDF), TNF-α, IL-4, and IL-13 (8).

Nanotechnology in Corneal NV

Angiogenesis plays a crucial role in ocular diseases, such as wet age-related macular degeneration, proliferative diabetic retinopathy, and corneal NV (72). At present, treatments for corneal NV include eyedrops of corticosteroids and NSAIDs, photodynamic therapy, photocoagulation, and intravitreous injection of VEGF inhibitors (21). However, recurrent intravitreous injections come with certain side effects, such as bleeding, eye infection, tearing, and detachment of vitreous/retina, not to mention the substantial financial burden. Hence, prevention of angiogenesis in a site-specific manner with long-lasting action is necessary. Benefits of using nanoparticles in medical treatments include sustained and prolonged drug release, reduced toxicity, and ease of manufacturing/scale-up.

VEGF-A is a critical proangiogenic factor that participates in many diseases, such as cancer and macular degeneration (5). It is also the key factor resulting in corneal NV; therefore, knockdown of the VEGF-A-associated gene actually leads to substantially reduced NV. Although the recombinant anti-VEGF-A plasmid is capable of inhibiting angiogenesis in the cornea, the short half-life/ duration of the plasmid compromised the efficacy of the treatment. Gene delivery of the anti-VEGF-A plasmid using nontoxic, biocompatible, and biodegradable nanoparticles as the carriers has been broadly discussed during the past 20 years. Nanoparticles are nanostructured particles ranging mostly from 200 to 500 nm. Owing to their unique physical properties, nanoparticles can be taken up by cells through phagocytosis and release their payload in a prolonged, sustained manner.

Small hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn and can be utilized to silence target gene expression through RNA interference (RNAi). In most cases, the expression of shRNA in cells is typically carried out by delivery of plasmids using viral (or bacterial) vectors. Previously, plasmids that express shRNA against VEGF-A were found to be capable of inhibiting and regressing corneal neovascularization (81). In addition, unlike small interfering RNAs (siRNAs), shRNAs are able to avoid immune activation due to their unique endogenous splicing property. Therefore, shRNAs present a relatively nonimmunogenic as well as a more specific, targeted antiangiogenic strategy, making it a potential translational tool for antiangiogenic gene therapy (72). Consequently, Qazi et al. manufactured shRNA-expressing plasmid-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles and tested their antiangiogenesis effects in mice with corneal NV. Encouragingly, those mice receiving the PLGA-shRNA-plasmid showed promising signs of inhibited corneal angiogenesis in significantly reduced VEGF-A mRNA and protein expression, compared to the animals receiving naked shRNA plasmid and empty PLGA nanoparticles. In addition, the significantly regressed mean fractional areas of corneal NV observed in the mice receiving PLGA-shRNA-plasmid 4 weeks postadministration further affirmed the improved duration of such a PLGA nanoparticle-based formulation (72).

Nanotechnology in Corneal Transplantation

Corneal transplantation is one of the most common organ transplantation procedures in the world. However, the high graft rejection rate in certain high-risk patients results in repeated surgery and shortage of donor corneas. Presently, steroid therapy is generally adopted to reduce inflammation and graft rejection after the surgery. Cho et al. chose biodegradable, biocompatible PLGA nanoparticles as the delivery platform for Flt23k (anti-VEGF intraceptor) expressing plasmid. Flt23k is a well-studied recombinant construct of VEGF-binding domains 2 and 3 of VEGF receptor-1, coupled with the endoplasmic reticulum retention signal sequence: lysine–aspartic acid– glutamic acid–leucine (KDEL). Flt23k intraceptors have been proven to intracellularly bind to VEGF, which subsequently inhibits VEGF secretion. In Cho et al.'s study, a remarkable synergistic effect was observed in mice that received both flt23k-PLGA NPs and steroids (triamcinolone acetonide) after corneal transplant (11). Significant antiangiogenesis, antilymphangiogenesis, and increased survival rate of corneal grafts were observed in a murine model. This invaluable work further broadened the therapeutic benefits of Flt23k to corneal vascularization.

Rapamycin is a newer immunosuppressant, which is used to prevent rejection in human organ transplantation. However, the low solubility and bioavailability have hindered its application. Zhang et al. prepared rapamycin loaded poly(ε-caprolactone)-poly(ethyleneglycol)-poly(ε-caprolactone) (PCEC) nanoparticles by an emulsion evaporation method for potential corneal transplantation use. The PCEC encapsulated rapamycin exhibited more than 100-fold increased solubility, compared to native rapamycin. The developed rapamycin-PCEC nanoparticles did not produce cytotoxicity in various eye-related cell lines (dose ranging from 0.05 to 10 mg/ml). In addition, an in vitro release study showed sustained rapamycin release from PCEC nanoparticles over a period of 48 h. As a result, nanoparticle-encapsulated rapamycin might be a potential immunosuppressive agent in corneal transplantation by instillation administration in the future (109).

Nanotechnology in Anticorneal Fibrosis

Corneal injury could ultimately cause a scar through corneal fibrosis, which is characterized by the presence of myofibroblasts and improper deposition of extracellular matrix (ECM) components (33). Luo et al. fabricated targeted intraceptor nanoparticles (biodegradable) to reduce angiogenesis and fibrosis in both primate and murine macular degeneration models. They used an anti-VEGF intraceptor, Flt23K, a recombinant construct of VEGF that specifically binds to domains 2 and 3 of vascular endothelial growth factor receptor-1 (VEGFR-1, Flt-1) and couples with the endoplasmic reticulum (ER) retention signal sequence KDEL (47). With a single intravenous injection of these targeted nanoparticles, the encapsulated recombinant Flt23k intraceptor plasmids were able to home to the neovascular lesions in the retina and regress corneal NV (with a nearly 40% restoration of visual loss induced by corneal NV) in primate and murine AMD models.

BMPs are a large family of growth factors with more than 10 members, and it has been documented that the cornea expresses several BMPs and their receptors (88). In addition, it has been documented that endogenous BMP7 levels in the eye and other organs decline when exogenous BMP7 has been administered to attenuate fibrosis in adult animal models (36). Tandon and coworkers investigated the effect of gold nanoparticle-mediated BMP7 gene therapy on corneal fibrosis using an in vivo rabbit model of laser ablation-induced corneal fibrosis. They found that such a nanoparticle-based, localized BMP7 gene delivery in rabbit cornea modulated wound healing and inhibited fibrosis in vivo through a counter balancing TGF-β1-mediated pro-fibrotic Smad pathway (88).

Nanotechnology in Corneal Gene Therapy

Corneal gene therapy can possibly cure acquired and inherited corneal diseases that otherwise lead to blindness. However, even though many studies have shown that gene therapy using viral vectors may be safe and effective in animal models for certain ocular diseases, the use of viral vectors is still questionable for real clinical applications due to their viral nature (30). Hu and colleagues designed calcium phosphate nanoparticles for the transfection of corneal endothelial cells and evaluated their function in both cell culture and explanted organs. Calcium phosphate nanoparticles (CaP-NP) are chemically similar to mammalian bones and teeth, are biodegradable, and do not have inherent toxicity. Hu et al. fabricated CaP-NP and functionalized these nanoparticles with pcDNA3-EGFP and using different amounts of poly(ethylenimine) (PEI) to further stabilize the delivery system. After transfection to human and murine corneal endothelial cells, EGFP expression remained stable, and the corneal endothelial cells exhibited minimal proliferative capacity and very low apoptosis after transfection with CaP-NP. Hence, CaP-NP may be a potential platform for corneal gene transfection without tumorigenesis concern.

Klausner et al. prepared chitosan-DNA nanoparticles to encapsulate six different plasmids for corneal gene delivery and studied transgene expression in rat corneas after injection of oligomeric chitosan-DNA nanoparticles into rat corneal stroma (37). One day after injection of nanoparticles into rat corneas, chitosan-DNA nanoparticles presented substantially higher transfected gene expression, compared to the commercially available viral-based transfection vectors. Therefore, such a chitosan-DNA-based gene delivery platform may provide another solution for enhanced transgene delivery/expression against corneal disorders.

Nanotechnology in Corneal Regeneration

Corneal damage by infection, dystrophy, trauma, and graft failure may cause corneal opacification, visual impairment, and even blindness. Currently, despite the fact that cornea transplantation is readily available, the sources of ideal corneal tissues are still inadequate. Therefore, a rapid-forming, feasible, and easy scale-up alternative for corneal transplantation is important. Recently, Ma et al. adopted biocompatible/biodegradable PLGA as the scaffold in generating autologous rabbit adipose-derived stem cells (rASCs) for corneal transplantation. The PLGA scaffold provided a three-dimensional environment for rASC to grow, which helped achieve improvement in corneal stromal defects. Furthermore, the rASCs were successfully differentiated into functional keratocytes capable of expressing aldehyde-3-dehydrogenase1A1 after transplantation for up to 24 weeks (48). Alkali burn is another issue leading to extensive damage to the cornea and often causes permanent visual damage. However, the current strategy to treat post-alkali burn corneal damage rarely restores the transparency and culminates in corneal haze and opacity. Therefore, Chowdhury and his colleagues invented pirfenidone-encapsulated PLGA nanoparticles for post-alkali burn cornea healing. Interestingly, pirfenidone was found capable of inhibiting the increase in TGF-β-induced collagen I and α-SMA synthesis through corneal fibroblasts in a dose-dependent manner. In addition, after topical administration to a murine model, pirfenidone-loaded NPs showed remarkable reduced collagen I level, corneal haze, and shortened time for corneal reepithelialization after alkali burn (12).

In summary, reconstruction of ocular surface, suppression of corneal NV resulting from various corneal insults, and development of cell therapies for corneal diseases are the main issues that need resolution currently. Improvement of in vitro cell expansion with the assistance of biocompatible scaffold/nanoparticles can effectively bypass the problem of corneal donor shortage. In the near future, with the combination of stem cell applications, novel ocular drug delivery systems shall be capable of ameliorating the therapeutic effects of current medication in corneal regenerative medicine.

Footnotes

Acknowledgments

The authors declare no conflict of interest.

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Stem Cell Therapy for Corneal Regeneration Medicine and Contemporary Nanomedicine for Corneal Disorders

Abstract

Keywords

Introduction

Corneal Stem Cells, Limbal Stem Cell Deficiency, and Stem Cell Therapies

Limbal Stem Cells and Associated Genetic Expression

Limbal Stem Cell Deficiency (LSCD) and Various Associated Treatments

Stem Cell Therapies for Ocular Surface Diseases

Embryonic Stem Cells (ESCs), Adult Stem Cells, and Induced Pluripotent Stem Cells (iPSCs)

Differentiation of Pluripotent Stem Cells

Other Applications of Pluripotent Stem Cells

Corneal Stromal Stem Cells

Corneal Endothelial Transplant and Cell Therapy

Corneal Endothelial Diseases and Associated Modern Keratoplasty

In Vitro Expansion of Corneal Endothelial Cells

Endothelial Cell Transplant

Potential Corneal Endothelial and Trabecular Meshwork Stem Cells

Nanotechnology in Corneal Disease

Nanodrugs in Corneal Diseases

Corneal Neovascularization

Nanotechnology in Corneal NV

Nanotechnology in Corneal Transplantation

Nanotechnology in Anticorneal Fibrosis

Nanotechnology in Corneal Gene Therapy

Nanotechnology in Corneal Regeneration

Footnotes

Acknowledgments

References