PURPOSE. To investigate the phenotypic changes of mature corneal epithelial cells (MCECs) that cocultured with limbal niche cells (LNCs) in three-dimensional Matrigel (3D Matrigel) in vitro. METHODS. MCECs were isolated from central corneas, and limbal epithelial progenitor cells (LEPCs) were isolated from limbal segments with Dispase II. LNCs were isolated and cultured from limbal niche using the collagenase A digestion method and identified with PCK/VIM/CD90/CD105/SCF/PDGFR beta. MCECs were cultured on 3D Matrigel (50%, v/v) with or without LNCs for 10 days. Expression of CK12 and p63 alpha and clone formation test were used to compare the progenitor phenotypic changes for MCECs before and after induction using LEPCs as control. RESULTS. Homogeneous LNCs were isolated and identified as spindle shape and adherent to a plastic surface coated with 5% Matrigel. Double immunostaining of the fourth-passage LNCs was uniformly PCK-/VIM+/CD90(+)/CD105(+)/SCF+/PDGFR beta(+). Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) revealed the decrease of PCK expression from the second passage and elevation of Vim, CD90, CD105, SCF, and PDGFR beta transcripts from the third passage, and the transcription level of Vim, CD90, CD105, SCF, and PDGFR beta was elevated statistically in the fourth passage compared to the first passage (P < 0.01). Both immunofluorescence (IF) staining for cross section and cytospin cells demonstrated that MCECs expressed higher CK12 while lower p63 alpha than LEPCs (P < 0.01). Sphere growth formation was noticed as early as 24 hours in the MCEC + LNC group, 48 hours in the LEPC group, and 72 hours in the MCEC group. The diameters of the spheres were the biggest in the MCEC + LNC group (182.24 +/- 57.91 mu m), smaller in the LEPC group (125.71 +/- 41.20 mu m), and smallest in the MCEC group (109.39 +/- 34.85 mu m) by the end of the 10-day culture (P < 0.01). Double immunostaining with CK12/p63 alpha showed that cells in the sphere formed from MCECs expressed CK12 but not p63 alpha; in contrast, some cells in the MCEC + LNC group expressed CK12, but most of them expressed p63 alpha. RT-qPCR revealed a significant reduction of CK12 transcript but elevation of p63 alpha, Oct4, Nanog, Sox2, and SSEA4 (P < 0.05). Holoclone composed of cubic epithelial cells could be generated in the MCEC + LNC group but not in the other two groups. CONCLUSIONS. The data shows that human MCEC cell phenotype could be induced to the dedifferentiation stage when cocultured with LNCs in 3D Matrigel that simulated the microenvironment of limbal stem cells in vitro.