Human Corneal Limbus Research Articles

Cultivation and differentiation characteristics of human limbal progenitor cells

Background The aim of this study was to investigate whether limbal progenitor cells can be cultured, expanded and differentiated in vitro not only to enter corneal differentiation but also towards RPE (retinal pigment epithelium) characteristics. Methods A 3 mm broad strip of human corneoscleral limbal tissue was digested enzymatically and cells were set into cell culture. Differentiation status and characteristics, proliferation and phagocytotic activity were assessed by immunocytochemical staining in combination with digital and confocal microscopy. Results Immunocytological analysis revealed expression of Nestin and p63 marker suggesting progenitor cell properties. Mitotic activity was demonstrated by BrdU (bromodesoxyuridine) uptake. Upon consecutive passages, corneal differentiation markers were predominantly expressed. Phagocytotic activity was demonstrated via uptake of FITC (fluorescein isothiocyanate) labelled latex beads. RPE markers Bestrophin and Cytokeratin 8/18 as well as glial marker GFAP and neuronal marker MAP with respective controls were negative indicating no differentiation towards characteristics of retinal pigment epithelium or neural and glial lineage. Conclusions The results suggest that isolation and cultivation of proliferating and phagocytotic cells from the human corneal limbus was achieved which showed characteristics of both progenitor and differentiated corneal cells. No evidence was found for the hypothesis of spontaneous differentiation potential towards RPE lineage or neuronal characteristics, providing evidence of the inherent directional capacity of limbal progenitor cells.

Characterization of the Limbal Epithelial Stem Cell Niche: Novel Imaging Techniques Permit In Vivo Observation and Targeted Biopsy of Limbal Epithelial Stem Cells

It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State-of-the-art imaging techniques were used to construct a three-dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63alpha and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies. Disclosure of potential conflicts of interest is found at the end of this article.

Melanocytes in the corneal limbus interact with K19-positive basal epithelial cells

The human corneal limbus is identified by the distinct features of the palisades of Vogt (POV), which contain pigment granules that are aligned with the microplicae of the epithelium. Although it is presumed that pigments are produced by melanocytes, the characterization of melanocytes in the limbus has not been clearly documented. We examined human limbal tissues by whole mounts and serial histological sections to localize epithelial cells containing melanin granules. Most of the pigmented cells observed by immunohistochemistry were K19 (+) cells in the basal limbal epithelium. A superimposed image revealed that melanin granules were oriented towards the apex of each K19 (+) cell, acting as a pigmented cap facing the ocular surface. Melanocytes were identified by MART1, an antigen specific to melanocyte-lineage cells. Melanocytes were shown to exist as sporadic cells with dendritic processes that extend to surrounding epithelial cells. Melanocytes were also found in light-pigmented donor tissue when visualized by the tyrosinase assay using the enzyme substrate DOPA. Since tyrosinase activity was not found in epithelial cells, the production of melanin is exclusively the role of melanocytes that comprised 5·3±2·7% of the total cells in cytospin samples ( N=3). Melanocytes and K19 (+) epithelial cells may form a functional network similar to the melanin unit of the skin.

DOUBLE NERVE SUPPLY OF KRAUSE CORPUSCLES AT THE HUMAN CORNEAL LIMBUS.

The Krause corpuscles of the conjunctiva and corneal limbus are well known to morphologists and physiologists as sensory receptors for the sensation of cold. 1,2 They are formed by relatively thick medullated nerve fibers originating in the ring-shaped paramarginal nerve plexus of Attias. 3 These enter the ovoid end-bulbs at their hilum and form a complex entanglement of terminal nerves within. The question whether the Krause end-bulbs have a connective tissue capsule of their own or not and whether the nuclei found accumulated around the entangled nerves are mesodermal or belong to Schwann cells 4,5 is not settled. It is known that one nerve fiber in its terminal course may form a number of Krause corpuscles hanging on them like a cherry on its stalk. Thus, many Krause bodies are seen to have an entrant and an emergent nerve. 6 Material and Method Tissue from the corneal limbus of 11