Sphere formation from corneal keratocytes and phenotype specific markers

The keratocytes are specialized mesenchymal cells that produce and maintain the extracellular matrix of the corneal stroma. With a typical dendritic and flattened appearance, these cells can morph into fibroblasts and myofibroblasts upon injury, and produce abnormal or fibrotic extracellular matrices detrimental to corneal transparency. Insights into mechanisms that regulate these phenotypic switches and optimal culture conditions that preserve the keratocyte phenotype are important for tissue engineering of the corneal stroma. Like other cell types with self-renewing capacity, keratocytes can form spheres in culture. Here we investigated human and bovine keratocytes with respect to their sphere forming capabilities, and sought to identify potentially distinguishing markers for the keratocyte and fibroblast phenotypes. Keratocytes, isolated from bovine and human corneas, cultured in serum-free medium supplemented with insulin, selenium and transferrin, assumed typical keratocyte morphology, converted to fibroblasts in serum-containing medium and reverted to keratocytes after serum-deprivation. The bovine keratocytes produced spheres under adherent or low attachment conditions, while the human keratocytes produced spheres under low attachment conditions only. The primary keratocytes and fibroblasts expressed vimentin, confirming their mesenchymal origin. Keratocan, considered to be a marker for keratocytes, was also detected in early passage bovine fibroblasts. BMP3 was expressed in keratocytes and keratocyte-derived spheres, while cadherin 5 in keratocytes only, suggesting these as potential keratocyte markers.     

A novel KERAmutation associated with autosomal recessive cornea plana

Purpose:To report a novel KERAmutation associated with autosomal recessive cornea plana in members of a nuclear family and to describe their ophthalmic phenotypes. Methods:Ophthalmic examination, biometry, and direct sequencing of KERA. Results:Five of the 6 siblings were affected and had small flat corneas, variable anterior chamber depths, and short axial lengths. The remaining brother and the 2 parents had normal ophthalmic examinations. Genetic testing revealed a novel homozygous nonsense mutation in exon 3 [937C>T] in the clinically affected individuals. The clinically unaffected parents were confirmed as carriers. The clinically unaffected sibling had no KERAmutation. This mutation leads to replacement of an arginine by a stop codon at position 313 of keratocan protein. Conclusions:This novel point mutation in KERAis the fourth thus far described. The ocular phenotype is characteristic of autosomal recessive cornea plana.     

Keratocan is Expressed by Osteoblasts and Can Modulate Osteogenic Differentiation

Keratocan is an extracellular matrix protein that belongs to the small leucine-rich proteoglycan family that also includes lumican, biglycan, decorin, mimecan, and fibromodulin. Members of this family are known to play a role in regulating cellular processes such as proliferation and modulation of osteoprogenitor lineage differentiation. The aims of this study were to evaluate the expression pattern of the keratocan within the osteoprogenitor lineage and to assess its role in regulating osteoblast maturation and function. Results from gene expression analyses of cells at different maturation stages within the osteoblast lineage indicate that keratocan is differentially expressed by osteoblasts and shows little or no expression by osteocytes. During primary osteoblast cultures, high keratocan mRNA expression was observed on day 14, whereas lower expression was detected at days 7 and 21. To assess the effects of keratocan on osteoprogenitor cell differentiation, we evaluated primary calvarial cell cultures from keratocan-deficient mice. The mineralization of calvarial osteoblast cultures derived from keratocan null (Kera–/–) mice was lower than in wild-type osteoblast cultures. Furthermore, analysis of RNA derived from Kera?/? calvarial cell cultures showed a reduction in the mature osteoblast differentiation markers, that is, bone sialoprotein and osteocalcin. In addition, we have evaluated the bone formation in keratocan-deficient mice. Histomorphometric analysis indicated that homozygous knockout mice have significantly decreased rates of bone formation and mineral apposition. Taken together, our results demonstrate the expression of keratocan by osteoblast lineage cells and its ability to modulate osteoblast function.