Human sclera maintains common characteristics with cartilage throughout evolution

Abstract

Background: The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm.

Methodology/principal findings: We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-betas and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human 'sclera'-derived cells can be considered chondrocytes when cultured ex vivo.

Conclusions/significance: Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia.

Figure 1. Proliferation of human ‘sclera’-derived cells.

A. Photograph of primary cultured human ‘sclera’-derived cells by phase-contrast microscope. B. Growth curve of cultured human ‘sclera’-derived cells. Vertical axis indicates population doublings (PD) and horizontal axis indicates days after innoculation of human ‘sclera’-derived cells.

Figure 2. Global gene expression analysis of cultured human cells.

A. Three-dimensional representation of PCA of gene expression levels (Human Genome U133 Plus 2.0: 54,675 probes). The gene expression data from scleral cells following one passage from the primary cultured cells (equivalent to appropriately 4 PDs) were used for PCA. Sclera and cartilage are positioned closely adjacent (shown in circle). B. Hierarchical clustering analysis based on the expression of all genes (Human Genome U133 Plus 2.0: 54,675 probes, NIA Array Analysis) shows similarity between scleral cells and chondrocytes. C. PCA of the cartilage-associated gene expression (Table S1). Sclera, cartilage, synovium, and joint fluid are positioned closely adjacent (shown in circle). D. Hierarchical clustering analysis based on expression levels of the cartilage-associated genes (NIA Array Analysis). Sclera, cartilage, synovium, and joint fluid are categorized into the same group. E. Hierarchical clustering analysis (TIGR MeV, see the Materials & Methods) with the heat map, based on expression levels of the cartilage-associated genes. Each row represents a gene; each column represents a cell population. Sclera, cartilage, synovium, and joint fluid are categorized into the same group. Cells derived from cartilage, synovium, and joint fluid are capable of generating cartilage in vivo , .

Figure 3. Chondrogenesis of human ‘sclera’-derived cells.

A. In vitro chondrogenesis. ‘Sclera’-derived cells were centrifuged to make a pellet and cultured in chondrogenic medium for 4 weeks. Macroscopic feature is shown. B. Histological section of a pellet by micromass culture in a chondrogenic medium stained with alcian blue. Bar: 100 µm. C. Reverse transcriptase-PCR for cartilage-associated genes. Total RNAs were prepared from scleral cells at passage 0, at 10 population doublings, after in vitro chondrogenic induction, and normal cartilage as a positive control. D. Histological sections 4 weeks after transplantation of human scleral cells into cartilage defect of the knee in a rat. (a) Toluidin blue staining. (b) Immunohistochemistry. Human scleral cells were labeled with DiI (red). Nuclei were stained with DAPI (blue). Type II collagen was shown as green.

Figure 4. The distribution of scleral cartilage in vertebrates.

The chondrogenic nature of the sclera is conserved across species. The figure is modified from Franz-Odendaal, TA, et al., 2006 . Species that have cartilage in the sclera are underlined; species with either absence or presence of cartilage in the sclera, depending on family, are dot-underlined; species without cartilage in the sclera are non-underlined.