TY - JOUR
T1 - Limbal Niche Cells and Three-Dimensional Matrigel-Induced Dedifferentiation of Mature Corneal Epithelial Cells
AU - Zhu, Hui
AU - Wang, Wei
AU - Tan, Yongyao
AU - Su, Guanyu
AU - Xu, Lingjuan
AU - Jiang, Meng lin
AU - Li, Shen
AU - Meir, Yaa Jyuhn James
AU - Wang, Yunming
AU - Li, Guigang
AU - Zhou, Huamin
N1 - Publisher Copyright:
© 2022 Association for Research in Vision and Ophthalmology Inc.. All rights reserved.
PY - 2022/5
Y1 - 2022/5
N2 - 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/PDGFRβ. MCECs were cultured on 3D Matrigel (50%, v/v) with or without LNCs for 10 days. Expression of CK12 and p63α 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 fourthpassage LNCs was uniformly PCK-/VIM+/CD90+/CD105+/SCF+/PDGFRβ +. 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 PDGFRβ transcripts from the third passage, and the transcription level of Vim, CD90, CD105, SCF, and PDGFRβ 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 p63α 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 μm), smaller in the LEPC group (125.71 ± 41.20 μm), and smallest in the MCEC group (109.39 ± 34.85 μm) by the end of the 10-day culture (P < 0.01). Double immunostaining with CK12/p63α showed that cells in the sphere formed from MCECs expressed CK12 but not p63α; in contrast, some cells in the MCEC + LNC group expressed CK12, but most of them expressed p63α. RT-qPCR revealed a significant reduction of CK12 transcript but elevation of p63α, 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.
AB - 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/PDGFRβ. MCECs were cultured on 3D Matrigel (50%, v/v) with or without LNCs for 10 days. Expression of CK12 and p63α 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 fourthpassage LNCs was uniformly PCK-/VIM+/CD90+/CD105+/SCF+/PDGFRβ +. 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 PDGFRβ transcripts from the third passage, and the transcription level of Vim, CD90, CD105, SCF, and PDGFRβ 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 p63α 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 μm), smaller in the LEPC group (125.71 ± 41.20 μm), and smallest in the MCEC group (109.39 ± 34.85 μm) by the end of the 10-day culture (P < 0.01). Double immunostaining with CK12/p63α showed that cells in the sphere formed from MCECs expressed CK12 but not p63α; in contrast, some cells in the MCEC + LNC group expressed CK12, but most of them expressed p63α. RT-qPCR revealed a significant reduction of CK12 transcript but elevation of p63α, 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.
KW - dedifferentiation
KW - limbal niche cell
KW - mature corneal epithelial cell
UR - https://www.scopus.com/pages/publications/85129781558
U2 - 10.1167/iovs.63.5.1
DO - 10.1167/iovs.63.5.1
M3 - 文章
C2 - 35499835
AN - SCOPUS:85129781558
SN - 0146-0404
VL - 63
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 5
ER -
