Dynamic spatiotemporal expression pattern of limbal stem cell putative biomarkers during mouse development.

Limbal stem cells (LSCs), a subpopulation of limbal epithelial basal cells, are crucial to the homeostasis and wound healing of corneal epithelium. The identification and isolation of LSCs remains a challenge due to lack of specific LSCs biomarkers. In this study, Haematoxylin-eosin (HE), 4', 6-diamidino-2-phenylindole (DAPI), and immunohistochemistry (IHC) stains were performed on the pre- and post-natal limbus tissues of mice which has the advantage of more controllable in term of sampling age relative to human origin. By morphological analysis, we supported that there is an absence of the Palisades of Vogt (POV) in the mouse. The development of prenatal and neonatal cornea was dominated by its stroma, whereas after eyelids opened at P14, the corneal epithelial cells (CECs) quickly go stratification in response to the liquid-air interface. Based on IHC staining, we found that the expression of LSCs putative biomarkers in limbal epithelial basal cells appeared in chronological order as follows: Vim = p63 > CK14 > CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells were weakened in chronological order as follows: Vim > p63 > CK15 > CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others putative molecular markers of LSCs, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.

Morphology of the extraocular muscles (m. bulbi) in the pre-hatchling and post-hatchling african black ostriches (struthio camelus domesticus L., 1758) (Aves: Struthioniformes).

The aim of the study was to describe the morphology and the development of the extraocular muscles (EOMs) in the pre-hatchling and post-hatchling African black ostrich. The study involved 50 birds aged between 28 days and 3 years. The EOMs were analyzed morphologically with respect to the location and length of the straight and oblique muscles and the third eyelid muscles, the length and breadth of their tendons as well as the distance and shape of the muscle tendon insertions at the corneal limbus. A histological and histometric analysis were also carried out. The greatest increase in the length of the EOMs was noted in groups III-V. A marked increase in the length of the tendons of the dorsal straight muscle was found in groups II and III, in the tendons of the nasal straight muscle in groups IV and V, in the tendons of the dorsal oblique muscle in groups III to V and in the tendons of the ventral oblique muscle in groups IV and V. There was a significant increase in the breadth of the dorsal straight and ventral oblique muscle tendons in groups IV and V and the tendons of the pyramidal muscle in groups III and V. The distance of the distal insertion of the tendon at the corneal limbus increased steadily with age in all the examined groups. The number of fascicles and muscle fibres, their diameter and length in all the studied EOMs were different in the different groups.

Down-regulation of Notch signaling during corneal epithelial proliferation.

PURPOSE: We evaluated the expression and activation of Notch pathway genes in the adult human and murine corneal epithelium during proliferation. METHODS: The expression of Notch pathway genes in the limbal and central human corneal epithelium was compared by reverse transcription polymerase chain reaction (RT-PCR). Their expression pattern was examined by immunofluorescence and in situ hybridization. The temporal expression of Notch1 during murine wound healing was assessed by RT-PCR. Notch activity was determined using western blot for the Notch intracellular domain (NotchIC). The expression of Hes1 was evaluated in cell culture. RESULTS: The expression of Notch1 and Jagged1 was higher in the human limbal epithelium while the expression of Hes1 and Hes5 was higher in the central cornea. Expression of Notch1, Jagged1, and Hes1 was found predominantly in the basal and immediate suprabasal cells. During neonatal corneal development, NotchIC was detected in occasional cells at P10 while at P15 and P90, it was found in the basal and early suprabasal layers. NotchIC was found to be lower in the limbal compared to central corneal epithelium. The expression of Notch1 was lower at 24 h post-wounding but was completely restored in six days. The levels of NotchIC were decreased at 24 h post-wounding and after application of topical phorbol myristate. In vitro, the expression of Hes1 was higher in confluent cells maintained under high calcium conditions. CONCLUSIONS: The inverse correlation between Notch signaling and the proliferative status of the corneal epithelium is consistent with the idea that Notch plays a role in corneal epithelial differentiation.

Immunohistochemical markers for corneal stem cells in the early developing human eye.

The corneal epithelium is continuously being renewed. Differentiated epithelial cells originate from limbal stem cells (LSCs) located in the periphery of the cornea, the corneoscleral limbus. We have recently identified superoxide dismutase 2 (SOD2) and cytokeratin (CK) 15 as limbal basal cell markers and potential markers for LSCs and early transient amplifying cells in human adults. In this study, we describe the development of the ectodermally derived LSCs and the mesodermally derived niche cells from the time at which the cornea is defined (week 6) until the formation of the early limbal niche (week 14) in human embryos and fetuses. The expression of SOD2 and CK15 was investigated together with other recently identified limbal proteins. Previously suggested LSC and differentiation markers (PAX6, aquaporin-1 and nestin) were also investigated. Both SOD2 and CK15 were present in the corneal epithelium from week 6. However, in week 14 they were predominantly expressed in the limbal epithelium. Both proteins were expressed already from week 7 in a stromal triangular region from which the early mesodermal limbal niche most likely originates. PAX6 was expressed in both ectodermally and mesodermally derived parts of the limbal niche, underscoring the importance of PAX6 in niche formation.

The phenotype of limbal epithelial stem cells.

PURPOSE: The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. METHODS: RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes. RESULTS: Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes. CONCLUSIONS: For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.

Ocular surface epithelial and stem cell development.

Phenotypic features and developmental events involved in the genesis of the limbo-corneal and conjunctival epithelia are described. Together, these two epithelia define the ocular surface. They derive from a small cohort of optic vesicle-induced PAX6+ head ectodermal cells that remain on the surface following lens vesicle formation by the main PAX6+ cell cohort. Both epithelia are stratified, and display wet, non-keratinizing phenotypes. The most significant spatial feature of the limbo-corneal epithelium is the segregation of its supporting stem and early precursor cells to the limbus, the outer vascularized rim separating the cornea from the conjunctiva. These stem cells express ABCG2, a xenobiotic transporter present in stem cells from other organs. ABCG2 transport activity excludes the DNA dye Hoechst 33342, allowing the isolation of the ocular stem cells by flow cytometry, as a unique cohort known as a side 'side population'. Limbal stem cells do not form gap junctions and exist as metabolically isolated entities. Tracking of expression changes in Cx43, the main gap junction protein expressed in both the pre-epithelial ectoderm and in the mature central corneal epithelium, indicates that a limbal stem cell phenotype starts developing very soon after lens vesicle invagination, in advance of the appearance of any recognizable anatomical sub-epithelial limbal feature. Differences in Cx43 expression also reveal the very early nature of the divergence in limbo-corneal and conjunctival lineages. The putative involvement of several early genes, including gradients of PAX6 and differences in expression patterns for members of the Id or msh gene expression regulators are reviewed.

Functional reconstruction of rabbit corneal epithelium by human limbal cells cultured on amniotic membrane.

PURPOSE: To investigate the phenotype of fetal and adult human limbal cells cultured on human amniotic membrane and the ability of cultured adult human limbal cells to repair limbal stem cell deficiency in a rabbit model. METHODS: Human adult and fetal limbal cells were isolated and cultured either on plastic plates or on human amniotic membrane. Connexin43, p63, and keratins 3 and 12 (K3 and K12) were detected by immunofluorescence and RT-PCR. Limbal stem cell deficiency was established in rabbits using chemical ablation and mechanical debridement. Cultured adult human limbal cells were transplanted onto rabbit corneas one month after injury, then fixed and imbedded in paraffin forty days later. Immunofluorescent staining of human-nuclear antigen, p63, K3, and connexin43 identified human-specific cells, progenitor cells, and differentiated corneal epithelial cells, respectively. RESULTS: Adult and fetal cultured limbal cells appeared similar in morphology. RT-PCR results showed that cells cultured from the human adult and fetal limbal area expressed both p63 and K12, whereas cells from central adult epithelium expressed K12 only. Immunofluorescent staining showed that more cells were p63 positive when cultured on human amniotic membrane than on plastic. Double staining for p63 and connexin43 showed some p63-positive cells co-expressing connexin43. After transplantation of adult human limbal cells cultured on human amniotic membrane, injured rabbit corneas were completely reconstructed exhibiting epithelial integrity, improved corneal clarity, and little or no neovascularization. The majority of repopulated epithelial cells expressed anti-human nuclear antibody. Cells expressing p63 occurred throughout the new epithelium. CONCLUSIONS: During healing, expression of p63 is not limited to epithelial stem cells but may also mark transient amplifying progenitor cells. Culture on human amniotic membrane suppresses differentiation of limbal epithelial cells and promotes the proliferation of p63 expressing cells. Amniotic membrane-cultured human limbal cells fully reconstructed rabbit corneas having limbal stem cell deficiency, with human cells providing most of the cells of the new epithelium. Expression p63 is distributed throughout the reconstructed tissue.