An Insight into the Difficulties in the Discovery of Specific Biomarkers of Limbal Stem Cells

Abstract

Keeping the integrity and transparency of the cornea is the most important issue to ensure normal vision. There are more than 10 million patients going blind due to the cornea diseases worldwide. One of the effective ways to cure corneal diseases is corneal transplantation. Currently, donations are the main source of corneas for transplantation, but immune rejection and a shortage of donor corneas are still serious problems. Graft rejection could cause transplanted cornea opacity to fail. Therefore, bioengineer-based corneas become a new source for corneal transplantation. Limbal stem cells (LSCs) are located at the basal layer in the epithelial palisades of Vogt, which serve a homeostatic function for the cornea epithelium and repair the damaged cornea. LSC-based transplantation is one of the hot topics currently. Clinical data showed that the ratio of LSCs to total candidate cells for a transplantation has a significant impact on the effectiveness of the transplantation. It indicates that it is very important to accurately identify the LSCs. To date, several putative biomarkers of LSCs have been widely reported, whereas their specificity is controversial. As reported, the identification of LSCs is based on the characteristics of stem cells, such as a nuclear-to-cytoplasm ratio (N/C) ≥ 0.7, label-retaining, and side population (SP) phenotype. Here, we review recently published data to provide an insight into the circumstances in the study of LSC biomarkers. The particularities of limbus anatomy and histochemistry, the limits of the current technology level for LSC isolation, the heterogeneity of LSCs and the influence of enzyme digestion are discussed. Practical approaches are proposed in order to overcome the difficulties in basic and applied research for LSC-specific biomarkers.

Process Development and Safety Evaluation of ABCB5+ Limbal Stem Cells as Advanced-therapy Medicinal Product to Treat Limbal Stem Cell Deficiency

“…Speci cally, we detected heterogenous expression of the wellrecognized LSC marker p63 [42] and homogenous expression of PAX6, which has been shown to play a critical role in limbal stem cell fate determination [51][52][53]. The absence of the lament proteins CK3/12, which are, due to their speci c expression in corneal epithelial cells and limbal suprabasal but not basal cells, regarded as markers of corneal epithelial differentiation [17,54,55], indicates that the cultivated LSCs have maintained their undifferentiated nature throughout the expansion process. Slight expression of the conjunctival epithelium marker CK19 and of vimentin is in line with reports that have detected these proteins, although inconsistently and not speci cally, in LSCs [17,55,56].…”

“…The absence of the lament proteins CK3/12, which are, due to their speci c expression in corneal epithelial cells and limbal suprabasal but not basal cells, regarded as markers of corneal epithelial differentiation [17,54,55], indicates that the cultivated LSCs have maintained their undifferentiated nature throughout the expansion process. Slight expression of the conjunctival epithelium marker CK19 and of vimentin is in line with reports that have detected these proteins, although inconsistently and not speci cally, in LSCs [17,55,56]. In contrast, the slight positivity for the gap junction protein connexin 43 was unexpected, as this protein was, similar to CK3/12, suggested as a putative negative biomarker of LSCs [57].…”

“…As the limbal region also contains Langerhans cells [17,66,67], which have been thought to be associated with graft failure in corneal transplantation [67], we needed to ensure that the nal drug product does not contain Langerhans cells either. One may nd it surprising that we could not detect any positivity for CD1a already in the corneal rims that served as starting tissue.…”

“…However, as LSCs comprise only a small population among heterogenous cell populations present in the limbus [16,17] and transplantation success highly depends on the percentage of LSCs within the transplanted cells [18], a major challenge in the further development of transplantation techniques has remained: the prospective identi cation of LSCs, which would permit enrichment of the stem cell content of the transplant. Over decades, LSCs could only be identi ed retrospectively by indirect or functional characteristics including label retention, lack of expression of corneal differentiation markers such as cytokeratin (CK)3, CK12 and CK19, ability to generate holoclones, and corneal epithelial regeneration capacity after transplantation [19].…”

Differentiation Induction of Human Stem Cells for Corneal Epithelial Regeneration

“…Connexin 43 (Cx43), a gap junction protein which is acquired during corneal epithelial differentiation, is expressed in the basal layer of the corneal epithelium but it is not expressed in the limbus [ 35 ]. Limbal epithelial cells express CK5, CK14 [ 54 ], and some putative stem cell markers such as tumor protein (p63), ATP-binding cassette transporter G2 (ABCG2), and CK19. These stem cells markers of limbal epithelial cells will gradually decrease and finally disappear when they become transient amplifying cells and migrate to the central cornea [ 55 ].…”