Sphere formation from corneal keratocytes and phenotype specific markers

Abstract

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 phenotype. 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.

Transforming Growth Factor β and Insulin Signal Changes in Stromal Fibroblasts of Individual Keratoconus Patients

“…Similarly, we recently showed that primary cells extracted from donor human corneas could be plated overnight in growth factor and serum-free DMEM: F12 for attachment and subsequently grown in the presence of ITS and phosphoascorbic acid expressed keratocan [32]. However, when we used this approach on freshly isolated stromal cells from keratoconus corneas, cell death was unacceptably high at the early stages and ultimately led to poor total cell yield.…”

Differentiation of Human Adipose-derived Stem Cells along the Keratocyte Lineage In vitro

“…These cells did not express α-SMA (Figure 1B). CDH5 was selected as a specific keratocyte marker since Scott et al [13] showed CDH5 was expressed only by keratocytes not by fibroblasts. Human lung fibroblasts treated by transforming growth factor beta (TGF-β) were used as positive control for α-SMA.…”

“…Thereafter, stromal cells were cultured in keratocyte growth medium (KGM) containing DMEM/F12 (Invitrogen-Gibco) with 10 ng/ml fibroblast growth factor 2(FGF2, Sigma-Aldrich), 1X insulin, transferrin, selenium (ITS, Invitrogen), 1 mM ascorbic acid-2-phosphate (Sigma-Aldrich), and 100 unit/ml penicillin (GIBCO), 100 μg/mL streptomycin (GIBCO). The cells were incubated at 37°C with 5% humidified CO 2 , and the medium was changed every 3~4 days [13]. When 80%–90% confluence achieved, they were trypsinized, subcultured and used for co-culture as described below.…”

Three-Dimensional Cell Culture Environment Promotes Partial Recovery of the Native Corneal Keratocyte Phenotype from a Subcultured Population

“…This finding is consistent with the plasticity observed in serum-free reversion of fibrotic corneal cells. 34 Thus, the addition of additional TGF-b would cause any cells that had begun to exhibit the native phenotype to once again revert. Cell reversion from a subcultured population is crucial to the success of a TE cornea and to making TE corneas widely available.…”