Recreating the Human Limbal Epithelial Stem Cell Niche with Bioengineered Limbal Crypts.

PURPOSE

Creation of an in vitro model incorporating specific features that characterize a particular stem niche would allow researchers to study stem cell behavior in a more physiological environment.

MATERIALS AND METHODS

We have developed a tissue engineering process (RAFT) that rapidly and reliably creates bioengineered limbal crypts (BLCs) in the surface of collagen-based tissue equivalents (TEs). These BLCs mimic the three-dimensional topography of the limbal crypts (LCs), located in the limbal region of the human cornea, which are home to a population of limbal epithelial stem cells (LESCs).

RESULTS

Human limbal epithelial (hLE) cells occupying our BLCs expressed putative LESC markers such as ΔNp63α and Bmi1 and produced basement membrane proteins such as laminin β1 and laminin γ3; expression patterns are very similar to those seen in native LCs. Human limbal stromal cells elongate and align along the edge of native LCs and in our RAFT TEs, human limbal fibroblasts (hLFs) also appeared to exhibit this alignment and elongation behavior in response to the BLC topography.

CONCLUSIONS

We have demonstrated that we can maintain an immature population of hLE cells and aligned stromal cells in our BLCs to mimic some elements of the complexity of the human LESC niche.