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.     

Profile of biological characterizations and clinical application of corneal stem/progenitor cells

Corneal stem/progenitor cells are typical adult stem/progenitor cells. The human cornea covers the front of the eyeball, which protects the eye from the outside environment while allowing vision. The location and function demand the cornea to maintain its transparency and to continuously renew its epithelial surface by replacing injured or aged cells through a rapid turnover process in which corneal stem/progenitor cells play an important role. Corneal stem/progenitor cells include mainly corneal epithelial stem cells, corneal endothelial cell progenitors and corneal stromal stem cells. Since the discovery of corneal epithelial stem cells (also known as limbal stem cells) in 1971, an increasing number of markers for corneal stem/progenitor cells have been proposed, but there is no consensus regarding the definitive markers for them. Therefore, the identification, isolation and cultivation of these cells remain challenging without a unified approach. In this review, we systematically introduce the profile of biological characterizations, such as anatomy, characteristics, isolation, cultivation and molecular markers, and clinical applications of the three categories of corneal stem/progenitor cells.     

兔角膜缘干细胞的研究进展

目前,角膜移植是临床上治疗角膜疾患的最有效途径,但供体角膜非常有限。新近兴起的干细胞技术,为组织工程角膜的研制和应用提供了契机。对于以角膜缘干细胞缺乏或功能障碍为特征的疾病也有治疗效果。采取角膜缘干细胞移植应是一种合理有效的治疗手段。本文主要介绍了兔角膜缘干细胞的体外分离、培养、鉴定及一些生长因子对其增殖的影响。     

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background

Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker.

Methodology/Principal Findings

K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker.

Conclusions/Significance

K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo.     

Limbal niche cells are a potent resource of adult mesenchymal progenitors

Limbal niche cells located in the limbal Palisades of Vogt are mesenchymal stem cells that reside next to limbal basal epithelial cells. Limbal niche cells are progenitors that express embryonic stem cell markers such as Nanog, Nestin, Oct4, Rex1, Sox2 and SSEA4, mesenchymal cell markers such as CD73, CD90 and CD105, and angiogenesis markers such as Flk‐1, CD31, CD34, VWF, PDGFRβ and α‐SMA, but negative for CD45. In addition, the stemness of limbal niche cells can be maintained during their cell culture in a three‐dimension environment. Furthermore, expanded limbal niche cells have the capability to undergo adipogenesis, chondrogenesis, osteogenesis and endogenesis in vitro, indicating that they are in fact a valuable resource of adult progenitors. Furthermore studies on how the limbal niche cells regulate the aforementioned stemness and corneal fate decision are warranted, as those investigations will shed new light on how mesenchymal progenitors reverse limbal stem cell deficiency and lead to new methods for limbal niche cell treatment.     

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.     

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.     

角膜新生血管的治疗新进展

角膜本身无血管,毛细血管网围绕角膜缘,如果血管超越角膜缘进入透明区即为病理性。角膜新生血管不是一种独立的角膜病,而是一种病理改变。由于维持角膜无血管的平衡因素被破坏,角膜缘的毛细血管侵入角膜周边部1-2 mm,即可视为角膜新生血管(CNV)形成1。随着它对感染的消除、创伤愈合、抑制免疫介导的角膜溶解有一定作用,但其结构和功能不完善,容易发生血浆渗漏,造成角膜水肿、脂质沉着以及激发的角膜瘢痕化,严重影响视力;也使角膜移植排斥反应的发生率大大增加。CNV的生长有利于病原微生物的清除和组织的修复,但严重影响了角膜的透明性,导致一系列的并发症,破坏眼球的完整性,目前国内外对治疗角膜新生血管的药物进行了大量的研究,希望通过不同的药物、不同的治疗方法解决这个大难题。     

Corneal stem cells and tissue engineering: Current advances and future perspectives

Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there are many challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.     

Rare corneal clones in mice suggest an age-related decrease of stem cell activity and support the limbal epithelial stem cell hypothesis

The anterior ocular surface comprises the cornea, conjunctiva and a narrow intermediate region called the limbus. It is widely accepted that the corneal epithelium is maintained by stem cells but different hypotheses propose that the stem cells that maintain the mouse corneal epithelium during normal homeostasis are located either in the basal limbal epithelium or throughout the basal corneal epithelium. There are no specific markers to help test these alternatives and new methods are required to distinguish between them. We observed that KRT5^LacZ/− transgenic mice produced rare β-galactosidase (β-gal)-positive radial stripes in the corneal epithelium. These stripes are likely to be clonal lineages of cells derived from stem cells, so they provide a lineage marker for actively proliferating stem cells. The distributions of the β-gal-positive radial stripes suggested they extended centripetally from the limbus, supporting the limbal epithelial stem cell (LESC) hypothesis. Stripe frequency declined between 15 and 30 weeks, which predicts a reduction in stem cell function with age. Pax6^+/−, KRT5^LacZ/− corneas had small patches rather than stripes, which confirms that corneal maintenance is abnormal in Pax6^+/− mice.     

Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells

Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function.     

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells

In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."     

Transdifferentiation of corneal epithelium: evidence for a linkage between the segregation of epidermal stem cells and the induction of hair follicles during embryogenesis

Corneal epithelium transdifferentiation into a hair-bearing epidermis provides a particularly useful system for studying the possibility that transient amplifying (TA) cells are able to activate different genetic programs in response to a change in their fibroblast environment, as well as to follow the different steps of rebuilding an epidermis from induced stem cells. Corneal stem and TA cells are found in different locations - stem cells at the periphery, in the limbus, and TA cells more central. Moreover, the TA cells already express the differentiating corneal-type keratin pair K3/K12, whereas the limbal keratinocytes express the basal keratin pair K5/K14. In contrast, suprabasal epidermal keratinocytes express keratin pair K1-2/K10, and basal keratinocytes the keratin pair K5/K14. The results of tissue recombination experiments show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. First, the cells located at the base of the corneal epithelium show a decrease in expression of K12 keratin, followed by an increase in K5 expression; they then proliferate and form hair follicles. The first K10 expressing cells appear at the junction of the new hair follicles and the covering corneal epithelium. Their expansion finally gives rise to epidermal strata, which displace the corneal suprabasal keratinocytes. Corneal TA cells can thus be reprogrammed to form epidermal cells, first by reverting to a basal epithelial-type, then to hair pegs and probably concomitantly to hair stem cells. This confirms the role of the hair as the main reservoir of epidermal stem cells and raises the question of the nature of the dermal messages which are both involved in hair induction and stem cell specification.     

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.     

Polysaccharide Hydrogel Combined with Mesenchymal Stem Cells Promotes the Healing of Corneal Alkali Burn in Rats

Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection of mesenchymal stem cells (MSCs) have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid and mesenchymal stem cells (MSCs) were derived from Sprague-Dawley rats. Supplementation of the polysaccharide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs application on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduction in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-β), antiangiogenic cytokine (TSP-1) and decrease those promoting inflammation (TNF-α), chemotaxis (MIP-1α and MCP-1) and angiogenesis (VEGF and MMP-2). This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treatments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder.     

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     

In search of markers for the stem cells of the corneal epithelium

The anterior one-fifth of the human eye is called the cornea. It consists of several specialized cell types that work together to give the cornea its unique optical properties. As a result of its smooth surface and clarity, light entering the cornea focuses on the neural retina allowing images to come into focus in the optical centres of the brain. When the cornea is not smooth or clear, vision is impaired. The surface of the cornea consists of a stratified squamous epithelium that must be continuously renewed. The cells that make up this outer covering come from an adult stem cell population located at the corneal periphery at a site called the corneal limbus. While engaging in the search for surface markers for corneal epithelial stem cells, vision scientists have obtained a better understanding of the healthy ocular surface. In this review, we summarize the current state of knowledge of the ocular surface and its adult stem cells, and analyse data as they now exist regarding putative corneal epithelial stem cell markers.