A Rapid Separation of Two Distinct Populations of Mouse Corneal Epithelial Cells with Limbal Stem Cell Characteristics by Centrifugation on Percoll Gradient

Krulová, Magdaléna; Pokorná, Kateřina; Lenčová, Anna; Frič, Jan; Zajı́cová, Alena; Filipec, Martin; Forrester, John V.; Holáň, Vladimı́r

doi:10.1167/iovs.08-1987

Cited by 23 publications

(25 citation statements)

References 30 publications

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“…The appearance of α-actin and vimentin in epithelial cells marks a pathological state of metaplasia. Our immunocytochemistry study on limbal epithelial cells showed staining for ABCG2, p63 and LGR5, which confirms previously published data (10,11) and confirms that they can be used as identifying markers for adult limbal stem cells. Our results show that a proportion of isolated human corneal epithelial cells possess limbal stem cell markers, indicating that these cells were still able to maintain the corneal limbal stem cell phenotype following cell isolation.…”

Section: Discussionsupporting

confidence: 91%

Molecular profile of organ culture-stored corneal epithelium: Lgr5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells

et al. 2012

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Abstract. Long-term preservation of corneal limbal epithelium may decrease its quality and change the molecular signature of the limbal epithelial stem cells. In this study we have investigated the molecular profile of isolated corneal epithelial cells that have been in storage for an extended time. Isolated cells were characterised by the expression profile of different cytokeratins and markers of squamous metaplasia (vimentin and α-actin). Furthermore, we examined global markers of adult stem cells including p63α and ABCG2 but also LGR5 as a novel stem cell marker. Immunocytochemical staining and PCR analysis of p63α, ABCG2 and LGR5 revealed the existence of side-population cells with a stem-cell phenotype and maintenance of corneal limbal stem cell properties. LGR5 expression can be related to cellular stemness and can be considered as a new phenotypic marker of residual human corneal limbal stem cells. However, the existence of CK10 together with co-expressed α-actin and vimentin suggests that the corneas investigated were under oxidative stress and showed evidence of squamous metaplasia.

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Section: Discussionsupporting

confidence: 91%

Molecular profile of organ culture-stored corneal epithelium: Lgr5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells

et al. 2012

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“…Conceptually, they may form a component of the molecular mechanisms by which long-lived stem cells reduce the potential for genomic damage over their extended lives, and their expression has been correlated with stem cell activity [90] . ABCG2 expression in the limbus is one such example and cells expressing this marker can be isolated as a "side population" by fluorescenceactivated cell sorting (FACS) [3,57,[91][92][93][94][95] . However, as noted above, some of the ABCG2-positive, labelretaining cells with a high nucleus to cytoplasm ratio cells in the rat limbus have been identified as Langerhans cells rather than epithelial stem cells [56] .…”

Section: Stem Cell Markers and Phenotypementioning

confidence: 99%

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

West¹,

Dora²,

Collinson

2015

WJSC

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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.

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“…A single-cell suspension from limbal tissues was obtained by enzyme digestion, as we have described [24]. In brief, limbal tissues (a narrow tissue between the cornea and the conjunctiva) from 10-12 BALB/c mice were cut out with scissors and subjected to 10 short (10 min each) trypsinization cycles.…”

Section: Preparation Of a Single-cell Suspension From Limbal Tissuementioning

confidence: 99%

The Key Role of Insulin-Like Growth Factor I in Limbal Stem Cell Differentiation and the Corneal Wound-Healing Process

Trošan

Svobodová

Chudíčková

et al. 2012

Stem Cells and Development

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Limbal stem cells (LSC), which reside in the basal layer of the limbus, are thought to be responsible for corneal epithelial healing after injury. When the cornea is damaged, LSC start to proliferate, differentiate, and migrate to the site of injury. To characterize the signaling molecules ensuring communication between the cornea and LSC, we established a mouse model of mechanical corneal damage. The central cornea or limbal tissue was excised at different time intervals after injury, and the expression of genes in the explants was determined. It was observed that a number of genes for growth and differentiation factors were significantly upregulated in the cornea rapidly after injury. The ability of these factors to regulate the differentiation and proliferation of limbal cells was tested. It was found that the insulin-like growth factor-I (IGF-I), which is rapidly overexpressed after injury, enhances the expression of IGF receptor in limbal cells and induces the differentiation of LSC into cells expressing the corneal cell marker, cytokeratin K12, without any effect on limbal cell proliferation. In contrast, the epidermal growth factor (EGF) and fibroblast growth factor-b (FGF-b), which are also produced by the damaged corneal epithelium, supported limbal cell proliferation without any effect on their differentiation. Other factors did not affect limbal cell differentiation or proliferation. Thus, IGF-I was identified as the main factor stimulating the expression of IGF receptors in limbal cells and inducing the differentiation of LSC into cells expressing corneal epithelial cell markers. The proliferation of these cells was supported by EGF and FGF.

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A Rapid Separation of Two Distinct Populations of Mouse Corneal Epithelial Cells with Limbal Stem Cell Characteristics by Centrifugation on Percoll Gradient

Cited by 23 publications

References 30 publications

Molecular profile of organ culture-stored corneal epithelium: Lgr5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells

Molecular profile of organ culture-stored corneal epithelium: Lgr5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

The Key Role of Insulin-Like Growth Factor I in Limbal Stem Cell Differentiation and the Corneal Wound-Healing Process

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