PURPOSE. Limbal stromal niche cells heterogeneously express embryonic stem cell (SC) markers. This study was conducted to isolate and expand them and to 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, or 3D Matrigel and were serially passaged in modified embryonic SC medium (MESCM), supplemented hormonal epithelial medium (SHEM), or Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (DF) before they were seeded in 3D Matrigel. Sphere growth was achieved by mixing expanded single cells with dispase-isolated epithelial cells in 3D Matrigel. 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 2D or coated Matrigel. Serial passages on coated Matrigel resulted in rapid expansion of spindle cells, of which the expression of ESC markers had declined but could be regained after reseeding in 3D Matrigel in MESCM but not in SHEM or DF. Resultant epithelial spheres mixed with spindle cells expanded in MESCM expressed more p63 alpha, less CK12, and more holoclones than those mixed with spindle cells expanded in DF. CONCLUSIONS. Limbal stromal niche cells expressing SC markers can be isolated and expanded to prevent differentiation and maintain clonal growth of limbal epithelial progenitors. (Invest Ophthalmol Vis Sci. 2012;53:279-286) DOI:10.1167/iovs.118441