Purpose: To investigate the potential of human corneal stromal stem cells to assume a keratocyte phenotype and to organize extracellular matrix (ECM) in vitro similar to corneal stromal tissue.
Methods: Human corneal stromal stem cells (hCSSC) were isolated as side population cells by flow cytometry. Cloned hCSSC were cultured as free-floating pellets in serum-free media for 3 weeks. Gene expression was examined using gene array, quantitative RT-PCR, immunostaining, and immunoblotting. Transmission electron microscopy showed collagen fibril size and alignment.
Results: Pellet cultures of hCSSC in serum-free media upregulated the expression of keratocyte-specific genes and secreted substantial ECM containing characteristic stromal components: keratocan, keratan sulfate, collagen I, collagen V, and collagen VI. Abundant connexin 43 and cadherin 11 in pellets demonstrated cell-cell junctions typical of keratocytes in vivo. Electron microscopy of the pellet cultures revealed abundant fibrillar collagen, some of which was aligned in parallel arrays similar to those of stromal lamellae. Gene array identified expression in pellets of several genes highly expressed by keratocytes. Transcripts for these keratocyte genes -- FLJ30046, KERA, ALDH3A1, CXADR, PTGDS, PDK4, MTAC2D1, F13A1 -- were increased by as much as 100-fold in pellets compared with hCSSC. Simultaneously, expression of stem cell genes BMI1, KIT, NOTCH1, SIX2, PAX6, ABCG2, SPAG10, and OSIL was reduced by a similar factor in pellets compared with hCSSC.
Conclusions: Scaffolding-free pellet culture of hCSSC induces keratocyte gene expression patterns in these cells and secretion of an organized stroma-like ECM. These cells offer a novel potential for corneal bioengineering.