Abstract
Purpose: :
Previously we developed a novel method of isolating limbal clusters containing entire limbal basal epithelial progenitors and their close associate niche cells expressing embryonic stem cell (ESC) markers. The purpose of this study is to further isolate and expand the close associated niche cells and demonstrate their phenotype is critical for supporting SCs.
Methods: :
1/12 piece of the human corneosclera ring was cut from 1 mm within and beyond the anatomic limbus and subjected to enzymatic digestion dispase (10 mg/ml) at 4 ºC or collagenase (1 mg/ml) at 37C for 16h. The resultant respective limbal epithelial sheets and clusters were further digested with 0.25% T/E at 37 ºC for 15 min to yield single cells that were seeded on coated, 2D, or 3D Matrigel and serially passaged in MESCM (modified embryonic SC medium), SHEM or DF (DMEM + 10% FBS) before being seeded in 3D Matrigel. Sphere growth was achieved by mixing the expanded isolated niche cells with dispase-isolated epithelial cells in 3D Matrigel culture. Expression of SC markers was analyzed by qRT-PCR, immunofluorescence staining, and western blot; SC clonal growth was measured on 3T3 feeder layers.
Results: :
Collagenase, but not dispase, isolated subjacent mesenchymal cells, of which the expression of Oct4, Sox2, Nanog, Rex1, SSEA4, N-Cadherin, and CD34 was promoted in MESCM more than SHEM or DF. Reunion of PCK+ and Vim+ cells generated spheres in 3D Matrigel, but spindle cells emerged on coated or 2D Matrigel. Serial passages on coated Matrigel resulted in rapid expansion of spindle cells, of which expression of ESC markers declined but could be regained upon reseeding in 3D Matrigel in MESCM but not SHEM or DF. Resultant epithelial spheres mixed with spindle cells expanded in MESCM expressed more p63α, less CK12, and more holoclones than those mixed with spindle cells expanded in DF.
Conclusions: :
These data collectively suggest that limbal stromal niche cells expressing SC markers can be isolated and expanded in MESCM and has the function in maintaining SC clonal growth and preventing limbal epithelial differentiation. Further optimization will help engineer a surgical graft containing limbal SCs and their niche cells for treating corneal blindness caused by limbal stem cell deficiency.
Keywords: cornea: basic science • cornea: stroma and keratocytes • cornea: epithelium