The Close Contact And Its Machanism Of Limbal Epithelial Stem Cells And Their Stromal Niche Cells

Part I Limbal Epithelial Stem/Progenitor Cells Attract Stromal Niche Cells by SDF-1/CXCR4Signaling to Prevent DifferentiationObjective:To investigate the function and mechanism of physical contact of limbal epithelial stem cells (SCs) and their native niche cells (NCs).Method:We have used collagenase digestion to isolate the limbal cells. Single cells were seeded on three-dimensional Matrigel in embryonic SC medium. In some cultures, AMD3100, CXCR4blocking antibody or CXCR7blocking antibody were added. Expression of SC markers was analyzed by qRT-PCR, immunofluorescence staining, and western blot; SC clonal growth was measured on3T3feeder layers.Results:Collagenase digestion isolated not only the limbal epithelial SCs but also subjacent mesenchymal cells. Inhibition of CXCR4by AMD3100or a blocking antibody to CXCR4but not CXCR7disrupted their reunion and yielded separate spheres with a reduced size, while resultant epithelial spheres exhibited more corneal differentiation and a notable loss of holoclones.Conclusions:For the first time, these results provide strong evidence supporting that limbal SC function depends on close physical association with their native NCs via SDF-1/CXCR4signaling. This novel in vitro model of sphere growth with NCs can be used for investigating how limbal SC self-renewal and fate decision might be regulated in the limbal niche. Part â…¡ Isolation and Expansion of Human Limbal Stromal Niche CellsObjective:The limbal stromal niche cells(NCs) heterogeneously express embryonic stem cell (SC) markers. This study is to isolate and expand them and prove that their phenotype is critical for supporting SCs.Methods:Human limbus was isolated by dispase or collagenase. Single cells were seeded on coated,2D or3D Matrigel and serially passaged in modified embryonic SC medium (MESCM), SHEM or DMEM+10%FBS (DF) before being seeded in3D Matrigel. Sphere growth was achieved by mixing expanded single cells with dispase-isolated epithelial cells in3D Matrigel. Expression of SC markers was analyzed by qRT-PCR, immunofluorescence staining, and western blot; SC clonal growth was measured on3T3feeder layers.Results:Collagenase, but not dispase, isolated subjacent mesenchymal cells, of which the expression of Oct4, Sox2, Nanog, Rex1, SSEA4, N-Cadherin, and CD34was promoted in MESCM more than SHEM or DF. Reunion of PCK+and Vim+cells generated spheres in3D Matrigel, but spindle cells emerged on2D or coated Matrigel. Serial passages on coated Matrigel resulted in rapid expansion of spindle cells, of which expression of ESC markers had declined but could be regained upon reseeding in3D Matrigel in MESCM but not SHEM or DF. Resultant epithelial spheres mixed with spindle cells expanded in MESCM expressed more p63a, less CK12, and more holoclones than those mixed with spindle cells expanded in DF.Conclusions:Limbal stromal NCs expressing SC markers can be isolated and expanded to prevent differentiation and maintain clonal growth of limbal epithelial progenitors.