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

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.