Keratocytes of the corneal stroma secrete a specialized extracellular matrix essential for vision. These quiescent cells exhibit limited capacity for self-renewal and after cell division become fibroblastic, secreting nontransparent tissue. This study sought to identify progenitor cells for human keratocytes. Near the corneal limbus, stromal cells expressed ABCG2, a protein present in many adult stem cells. The ABCG2-expressing cell population was isolated as a side population (SP) by cell sorting after exposure to Hoechst 33342 dye. The SP cells exhibited clonal growth and continued to express ABCG2 and also PAX6, product of a homeobox gene not expressed in adult keratocytes. Cloned SP cells cultured in medium with fibroblast growth factor-2 lost ABCG2 and PAX6 expression and upregulated several molecular markers of keratocytes, including keratocan, aldehyde dehydrogenase 3A1, and keratan sulfate. Cloned corneal SP cells under chondrogenic conditions produced matrix staining with toluidine blue and expressed cartilage-specific markers: collagen II, cartilage oligomatrix protein, and aggrecan. Exposure of cloned SP cells to neurogenic culture medium upregulated mRNA and protein for glial fibrillary acidic protein, neurofilament protein, and beta-tubulin II. These results demonstrate the presence of a population of cells in the human corneal stroma expressing stem cell markers and exhibiting multipotent differentiation potential. These appear to be the first human cells identified with keratocyte progenitor potential. Further analysis of these cells will aid elucidation of molecular mechanisms of corneal development, differentiation, and wound healing. These cells may be a resource for bioengineering of corneal stroma and for cell-based therapeutics.

https://stemcellsjournals.onlinelibrary.wiley.com/doi/pdf/10.1634/stemcells.2004-0256

Multipotent stem cells in human corneal stroma.

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene.

Multipotent Stem Cells in Human Corneal Stroma

Characterization and Expression of the Mouse Lumican Gene

The cornea is the outermost surface of the eye and the portal for light entry into the visual system. The strength and transparency of this organ rely on the highly organized extracellular matrix (ECM) of the corneal stroma, a tissue elaborated by unique neural crest-derived cells known as keratocytes. The stromal tissue is composed of 20 to 70 acellular collagenous lamellae of heterotypic types I and V fibrils with small uniform diameters, tightly packed in parallel orientation.1 This fibrillar collagen is embedded in a matrix of glycoproteins and proteoglycans that includes type VI nonfibrillar collagen and a unique family of keratan sulfate-containing proteoglycans essential for corneal transparency. The keratocytes, sandwiched between stromal lamellae, are the source of the molecular components of the stromal ECM and are largely quiescent in adult mammals. During wound healing, keratocytes become mitotic and motile, undergoing alteration of their ECM phenotype, depositing opaque scar tissue capable of causing permanent disruption of visual acuity. Keratoplasty using donated human tissue is currently the only effective procedure to correct visual impairment resulting from stromal scarring.

We recently identified a small population of cells in human corneal stroma with properties of adult stem cells.2 As do other adult stem cells, these cells express mRNA and protein for the multidrug transporter ABCG2 and can be expanded in culture through a number of population doublings without loss of this expression. These cells also express PAX6, a gene product present in embryonic and developing stromal cells but not in adult keratocytes.2 Like mesenchymal stem cells, corneal stromal stem cells (hCSSC) exhibit clonal growth and a potential for differentiation into multiple distinct tissue types.2 They express gene products characteristic of chondrocytes and neural cells when placed in culture environments known to elicit these phenotypes from other adult stem cell populations.2 As they differentiate, hCSSC lose ABCG2 and PAX6 expression. These stem cell markers were not regained when the differentiated cells were returned to stem cell maintenance culture conditions.2

The hCSSC expressed keratocan mRNA and protein as well as high molecular weight keratan sulfate when cultured in serum-free medium containing FGF2 and insulin. Keratocan glycanated with keratan sulfate is a unique product of the corneal stroma and is lost during in vitro expansion of cultured keratocytes.3,4 Secretion of this proteoglycan by cells derived from passaged hCSSC represents the first demonstration of induction of this keratocyte-specific molecule by passaged human cells in culture. Expression of these unique keratocyte products suggests hCSSC can adopt a keratocyte phenotype; however, in monolayer culture, these cells do not accumulate and organize the highly specialized ECM produced by stromal keratocytes in vivo. In the present study we examined the ability of three-dimensional culture conditions to induce elaboration of an organized stromal matrix by hCSSC. We also used gene array analyses to provide a more complete assessment of the gene expression phenotype of human keratocytes. These analyses yielded a panel of 18 genes providing an improved molecular definition of keratocyte phenotype. We found that culture of the hCSSC as a cell pellet in serum-free medium in the absence of rigid scaffolding or substratum induced an expression pattern of genes similar to that of keratocytes. Matrix expression and collagen organization in these pellets exhibited aspects of those seen in corneal stroma.

Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma

• Macular corneal dystrophy is an autosomal recessive disorder in which abnormal deposits in the corneal stroma have been identified. We examined the corneal buttons of 12 patients, who had clinical features of macular dystrophy, by histochemical staining, transmission electron microscopy, and immunohistochemical techniques. All corneas exhibited positive staining with Muller Mowry's colloidal iron. Using monoclonal antibodies 1/20/ 5-D-4, J-10, J-19, and J-36 that recognize specific sites on the sulfated keratan sulfate molecule, we stained corneal sections by an avidin-biotin-peroxidase complex method and identified two groups of macular corneal dystrophy. One group consisting of four corneas reacted positively with all four antibodies, and the other group consisting of eight corneas did not react with any of the antibodies used. These results confirmed those recently presented by Yang et al that there may be subgroups of macular dystrophy that can be identified by immunohistochemical methods. Also, serum levels of sulfated keratan sulfate were determined in seven patients. One patient who displayed a normal level of serum keratan sulfate had positive corneal immunoreactivity. Of the six patients who lacked serum keratan sulfate, four showed negative and two had positive corneal immunostaining, suggesting at least three sub-groups in the disease. An attempt was made to correlate the clinical features, histochemical-staining characteristics, and ultrastructural morphology with the immunoreactivity to keratan sulfate antibodies, but no correlations could be made.

E. L. Stock

Papers

Heterogeneity in Macular Corneal Dystrophy