A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology

Shaharuddin, Bakiah; Ahmad, Sajjad; Latar, Nani Harlina Md; Ali, Simi; Meeson, Annette

doi:10.5966/sctm.2016-0175

Cited by 14 publications

(11 citation statements)

References 17 publications

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“…The images were processed using Zen (blue edition) software from Carl Zeiss. In additional experiments, the ABCB5 antibody was validated against previously established ABCB5 monoclonal (clone 5H3C6) (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively, the ATP binding cassette subfamily B member 5 (ABCB5) surface protein, has been suggested as a putative hLESCs marker. Recent findings have shown that ABCB5 is critical for corneal epithelial homeostasis and repair and it is often co‐expressed with p63 in hLESCs both in situ and ex situ . However, many concerns were recently raised regarding the ability of p63, as well as of ABCB5 to accurately detect hLESCs .…”

Section: Introductionmentioning

confidence: 99%

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Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells

et al. 2018

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Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells

et al. 2018

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“…A small amount of LSCs are hidden among the LECs, which account for only 0.5–10% [ 18 ], resulting in the difficulties with isolating the LSCs with such low frequency from such complex limbal epithelium suspension. As reported, there were some traditional methods to isolate the LSCs such as gradient centrifugation [ 120 ], FACS [ 121 ], and magnetic-activated cell sorting (MACS) [ 64 ]. All of these methods are based on enzyme digestion, which potentially changes gene expression patterns and protein profiles of LSCs, and increases the risk of contamination of suspension due to complicated operations [ 122 , 123 , 124 ].…”

Section: Difficulties In the Study Of Lsc-specific Biomarkersmentioning

confidence: 99%

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

Guo

Zhang

Jia

et al. 2018

IJMS

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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.

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“…This discovery identified ABCB5 as the first molecular surface marker for prospective LSC enrichment by antibody-based cell sorting. Furthermore, Abcb5 gene loss of function in Abcb5 -knockout mice was associated with defective corneal differentiation and regeneration, indicating that ABCB5, beyond representing an LSC marker [ 24 – 27 ], is required for LSC function and corneal epithelial regeneration [ 23 ]. Purified CK12 − ABCB5 + LSCs could, in vitro, be induced to differentiate into CK12 + epithelial cells [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

Process development and safety evaluation of ABCB5+ limbal stem cells as advanced-therapy medicinal product to treat limbal stem cell deficiency

Norrick¹,

Esterlechner²,

Niebergall-Roth³

et al. 2021

Stem Cell Res Ther

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Background While therapeutic success of the limbal tissue or cell transplantation to treat severe cases of limbal stem cell (LSC) deficiency (LSCD) strongly depends on the percentage of LSCs within the transplanted cells, prospective LSC enrichment has been hampered by the intranuclear localization of the previously reported LSC marker p63. The recent identification of the ATP-binding cassette transporter ABCB5 as a plasma membrane-spanning marker of LSCs that are capable of restoring the cornea and the development of an antibody directed against an extracellular loop of the ABCB5 molecule stimulated us to develop a novel treatment strategy based on the utilization of in vitro expanded allogeneic ABCB5+ LSCs derived from human cadaveric limbal tissue. Methods We developed and validated a Good Manufacturing Practice- and European Pharmacopeia-conform production and quality-control process, by which ABCB5+ LSCs are derived from human corneal rims, expanded ex vivo, isolated as homogenous cell population, and manufactured as an advanced-therapy medicinal product (ATMP). This product was tested in a preclinical study program investigating the cells’ engraftment potential, biodistribution behavior, and safety. Results ABCB5+ LSCs were reliably expanded and manufactured as an ATMP that contains comparably high percentages of cells expressing transcription factors critical for LSC stemness maintenance (p63) and corneal epithelial differentiation (PAX6). Preclinical studies confirmed local engraftment potential of the cells and gave no signals of toxicity and tumorgenicity. These findings were sufficient for the product to be approved by the German Paul Ehrlich Institute and the U.S. Food & Drug Administration to be tested in an international multicenter phase I/IIa clinical trial (NCT03549299) to evaluate the safety and therapeutic efficacy in patients with LSCD. Conclusion Building upon these data in conjunction with the previously shown cornea-restoring capacity of human ABCB5+ LSCs in animal models of LSCD, we provide an advanced allogeneic LSC-based treatment strategy that shows promise for replenishment of the patient’s LSC pool, recreation of a functional barrier against invading conjunctival cells and restoration of a transparent, avascular cornea.

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A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology

Cited by 14 publications

References 17 publications

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells

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

Process development and safety evaluation of ABCB5+ limbal stem cells as advanced-therapy medicinal product to treat limbal stem cell deficiency

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