Single-cell RNA sequencing in cornea research: Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye, which acts as a protective barrier and is critical for clear and stable vision. Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells (LSCs), a cell population that resides at the limbus in a highly regulated niche. Dysfunction of LSCs or their niche can cause limbal stem cell deficiency, a disease that is manifested by failed epithelial wound healing or even blindness. Nevertheless, compared to stem cells in other tissues, little is known about the LSCs and their niche. With the advent of single-cell RNA sequencing, our understanding of LSC characteristics and their microenvironment has grown considerably. In this review, we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology, including the heterogeneity of the LSC population, novel LSC markers and regulation of the LSC niche, which will provide a reference for clinical issues such as corneal epithelial wound healing, ocular surface reconstruction and interventions for related diseases.     

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

Background  

The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.     

Enrichment of corneal epithelial stem/progenitor cells using cell surface markers, integrin alpha6 and CD71

Corneal epithelial stem cells are believed to reside in the basal layer of the limbal epithelium, but no definitive cell surface markers have been identified. For keratinocytes, stem/progenitor cells are known to be enriched by cell surface markers, integrin α₆ and CD71, as a minor subpopulation which shows high integrin α₆ and low CD71 expressions (α₆^bri/CD71^dim). In the present study, we investigated the possibility that corneal epithelial stem cells can be enriched by integrin α₆ and CD71. The α₆^bri/CD71^dim cells were separated by fluorescence-activated cell sorting, as a minor subpopulation of the limbal epithelial cells. They were enriched for relatively small cells, showing a higher clonogenic capacity and expression of stem cell markers, but a lower expression of differentiation markers, compared to other cell populations. The cells were localized immunohistochemically in the basal region of the limbal epithelium. These results indicate that the α₆^bri/CD71^dim subpopulation enriched corneal epithelial stem cells.     

Isoforms of p63 in corneal stem cells cultured on human amniotic membrane

The p63 gene supports stem cell proliferation and regulation in epithelial cells. In this study, corneal epithelial cells were cultured on human amniotic membrane (HAM) and investigated for p63 and its isoform genes. Human limbal biopsies obtained from cadaveric donor eyes were cultivated on intact and denuded HAM. Transactivation (TA) specific domain was positive in the limbal cells cultured over denuded HAM and negative on others. TAp63α,β,γ isoforms are negative in all the limbal cells cultured on intact, denuded and limbal tissues but not in cornel epithelial tissue. p63α isoform is present in all except on denuded HAM. αβ sharing region is not expressed only in cornel epithelial tissue. γ isoform is expressed in all the samples. ΔNp63α region is present in cells cultured over the intact HAM whereas it is negative on the cells cultured over the denuded HAM. The other isoforms such as ΔNp63β and ΔNp63γ are negative in all samples. The limbal cells cultured over the intact HAM were able to maintain high proliferative potential when compared to denuded HAM. Thus, p63 isoforms plays a biological function to retain the proliferative capacity of corneal epithelial cells and maintains the stemness when cultured on intact HAM.     

Lycopersicon esculentum lectin is a marker of transient amplifying cells in in vitro cultures of isolated limbal stem cells

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SCs) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SCs biology remains the ability to identify stem cells in situ and in vitro. To date, not so much markers exist for the identification of different phenotypes. CESCs (corneal epithelial stem cells) isolated from limbal biopsies were maintained in primary culture for 14 days and stained with Hoechst and a panel of FITC-conjugated lectins. All lectins, with the exception of Lycopersicon esculentum, labelled CESCs irrespective of the degree of differentiation. Lycopersicon esculentum, that binds N-acetylglucosamine oligomers, labelled intensely only the surface of TACs (single corneal epithelial stem cells better than colonial cells). These results suggest that Lycopersicon esculentum lectin is a useful and easy-to-use marker for the in vitro identification of TACs (transient amplifying cells) in cultures of isolated CESCs.     

Ophthalmic Applications of Preserved Human Amniotic Membrane: A Review of Current Indications

Preserved human amniotic membrane (AM) is currently being used for a wide spectrum of ocular surface disorders. The AM has a basement membrane, which promotes epithelial cell migration and adhesion. The presence of a unique avascular stromal matrix reduces inflammation, neovascularization and fibrosis. The basic tenets of amniotic membrane transplantation (AMT) are to promote re-epithelialization, to reconstruct the ocular surface and to provide symptomatic relief from surface aberrations. AMT is a useful technique for reconstruction of surface defects resulting from removal of surface tumors and symblephara. AMT has effectively restored a stable corneal epithelium in eyes with, persistent epithelial defects and corneal ulcers. In the setting of acute ocular burns and SJS, AMT has satisfactorily reduced scarring and inflammation. AMT alone may be an effective alternative for partial limbal stem cell deficiency. However remarkable improvements in surface stability have resulted from concurrent AMT and limbal stem cell transplantation, wherein the limbal grafts are obtained from the normal fellow eye, living relative or cadaveric eye. In severe or bilateral cases, well being of the donor eye is a major concern. Currently, the most unique application of preserved human AM in ophthalmology is its use as a substrate for ex-vivo expansion of corneal and conjunctival epithelium. In this novel technique of tissue engineering, epithelial stem cells can be safely harvested and expanded on denuded AM. The resultant composite cultured tissue has been successfully transplanted to restore vision, as well as the structure and function of damaged ocular surfaces.     

Niche regulation of corneal epithelial stem cells at the limbus

Among all adult somatic stem cells,those of the corneal epithelium are unique in their exclusive location in a definedlimbai structure termed Palisades of Vogt.As a result,surgical engraftment oflimbal epithelial stem cells with or withoutex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency.Neverthe-less,compared to other stem cell examples,relatively little is known about the limbal niche,which is believed to play apivotal role in regulating self-renewal and fate decision of limbal epithelial stem cells.This review summarizes relevantliterature and formulates several key questions to guide future research into better understanding of the pathogenesis oflimbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing onthe limbal niche.     

Pigment epithelium‐derived factor peptide promotes limbal stem cell proliferation through hedgehog pathway

Expansion of limbal epithelial stem cells (LSCs) is crucial for the success of limbal transplantation. Previous studies showed that pigment epithelium‐derived peptide (PEDF) short peptide 44‐mer could effectively expand LSCs and maintain them in a stem‐cell state, but the mechanism remained unclear. In the current study, we found that pharmacological inhibition of Sonic Hedgehog (SHh) activity reduced the LSC holoclone number and suppressed LSC proliferation in response to 44‐mer. In mice subjected to focal limbal injury, 44‐mer facilitated the restoration of the LSC population in damaged limbus, and such effect was impeded by the SHh or ATGL (a PEDF receptor) inhibitor. Furthermore, we showed that 44‐mer increased nuclear translocation of Gli1 and Gli3 in LSCs. Knockdown of Gli1 or Gli3 suppressed the ability of 44‐mer to induce cyclin D1 expression and LSC proliferation. In addition, ATGL inhibitor suppressed the 44‐mer‐induced phosphorylation of STAT3 at Tyr705 in LSC. Both inhibitors for ATGL and STAT3 attenuated 44‐mer‐induced SHh activation and LSC proliferation. In conclusion, our data demonstrate that SHh‐Gli pathway driven by ATGL/STAT3 signalling accounts for the 44‐mer‐mediated LSC proliferation.     

Therapeutic use of limbal stem cells

Stem cells are defined as relatively undifferentiated cells that have the capacity to generate more differentiated daughter cells. Limbal stem cells are responsible for epithelial tissue repair and regeneration throughout the life. Limbal stem cells have been localized to the Palisades of Vogt in the limbal region. Limbal stem cells have a higher proliferative potential compared to the cells of peripheral and central cornea. Limbal stem cells have the capacity to maintain normal corneal homeostasis. However, in some pathological states, such as chemical and thermal burns, Stevens-Johnson syndrome, and ocular pemphigoid limbal stem cells fail to maintain the corneal epithelial integrity. In such situations, limbal stem cell transplantation has been required as a therapeutic option. In unilateral disorders, the usual source of stem cells is the contralateral eyes, but if the disease is bilateral stem cell allografts have to be dissected from family members or cadaver eyes. The advent of ex vivo expansion of limbal stem cells from a small biopsy specimen has reduced the risk of limbal deficiency in the donor eye. Concomitant immunosuppressive therapy promotes donor-derived epithelial cell viability, but some evidences suggest that donor-derived epithelial stem cell viability is not sustained indefinitely. Thus, long-term follow-up studies are required to ascertain whether donor limbal stem cell survival or promotion of recolonization by resident recipient stem cells occurs in restored recipient epithelium. However, this is not an easy task since a definitive limbal stem cell marker has not been identified yet. This review will discuss the therapeutic usage of limbal stem cells in the corneal epithelial disorders.     

Limbal stem cells: Central concepts of corneal epithelial homeostasis

A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface     

Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.     

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell(LESC)hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient(or transit) amplifying cells(TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell(CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.     

Limbal stem cells, a review of their identification and culture for clinical use

The surface of the eye is covered by two distinct epithelial populations, the conjunctival and corneal epithelia. The stem cell population for the corneal epithelia has been found to be located at the area known as the limbus. This is a narrow ring of tissue at the transitional zone between the cornea and conjunctiva. This stem cell population is responsible for generating transient amplifying cells which are responsible for renewing the cornea epithelia. There are currently no definitive markers for the stem cell population in the limbus. Instead using morphological features, such as small cells with a high nucleus-to-cytoplasm ratio, in conjunction with the presence of certain markers e.g. ΔNP63α and the absence of others, e.g. the cytokeratin pair 3 & 12, are taken as being indicative of the stem cell population. Damage can occur to the corneal epithelium due to a number of causes including, Steven-Johnson syndrome, and chemical or thermal burns. This results in invasion of the cornea by the conjunctival epithelium resulting in impaired vision. In 1997 Pellegrini et al. (Lancet 349, 990) successfully used cells sheets from cultured limbal cells to successfully treat patients with corneal damage. Since then several other groups, have successfully treated patients, using similar methods.     

Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium

We have previously shown that a basic 64-kilodalton (no. 3 in the catalog of Moll et al.) and an acidic 55-kilodalton (no. 12) keratin are characteristic of suprabasal cell layers in cultured rabbit corneal epithelial colonies, and therefore may be regarded as markers for an advanced stage of corneal epithelial differentiation. Moreover, using an AE5 mouse monoclonal antibody, we showed that the 64-kilodalton keratin marker is expressed suprabasally in limbal epithelium but uniformly (basal layer included) in central corneal epithelium, suggesting that corneal basal cells are in a more differentiated state than limbal basal cells. In conjunction with previous data implicating the centripetal migration of corneal epithelial cells, our data support a model of corneal epithelial maturation in which corneal epithelial stem cells are located in the limbus, the transitional zone between the cornea and conjunctiva. In the present study, we analyzed the expression of the 64-kilodalton keratin in developing human corneal epithelium by immunohistochemical staining. At 8 weeks of gestation, the presumptive corneal epithelium is composed of a single layer of cuboidal cells with an overlying periderm; neither of these cell layers is AE5 positive. At 12-13 weeks of gestation, some superficial cells of the three- to four-layered epithelium become AE5 positive, providing the earliest sign of overt corneal epithelial differentiation. At 36 weeks, although the epithelium is morphologically mature (four to six layers), AE5 produces a suprabasal staining pattern, this being in contrast to the adult epithelium which exhibits uniform staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ex vivo expansion of limbal stem cells is affected by substrate properties

Limbal epithelial stem cells play a key role in the maintenance and regulation of the corneal surface. Damage or destruction of these cells results in vascularisation and corneal opacity. Subsequent limbal stem cell transplantation requires an ex vivo expansion step and preserving cells in an undifferentiated state remains vital. In this report we seek to control the phenotype of limbal epithelial stem cells by the novel application of compressed collagen substrates. We have characterised the mechanical and surface properties of conventional collagen gels using shear rheology and scanning electron microscopy. In doing so, we provide evidence to show that compressive load can improve the stiffness of collagen substrates. In addition Western blotting and immunohistochemistry display increased cytokeratin 3 (CK3) protein expression relating to limbal epithelial cell differentiation on stiff collagen substrates. Such gels with an elastic modulus of 2900 Pa supported a significantly higher number of cells than less stiff collagen gels (3 Pa). These findings have substantial influence in the development of ocular surface constructs or experimental models particularly in the fields of stem cell research, tissue engineering and regenerative medicine.