Arresting proliferation improves the cell identity of corneal endothelial cells in the New Zealand rabbit.
Mol Vis
; 25: 745-755, 2019.
Article
em En
| MEDLINE
| ID: mdl-31814700
Purpose: Corneal endothelium engineering aims to reduce the tissue shortage for corneal grafts. We investigated the impact of mitogenic and resting culture systems on the identity of corneal endothelial cells (CECs) for tissue engineering purposes. Methods: Rabbit CECs were cultured in growth factor-supplemented media (MitoM) until confluence. At the first passage, the CECs were divided into two populations: P1 remained cultured in MitoM, and P2 was cultured in a basal medium (RestM) for another passage. Morphologic changes in the CECs were analyzed, and RNA was isolated for transcriptome analysis. Quantitative PCR and immunocytochemistry validation of selected differentially expressed markers were performed. Results: The CECs in MitoM showed fibroblastic morphology, whereas the CECs in RestM exhibited polygonal morphology. Circularity analysis showed similar values in human (0.75±0.056), rabbit basal (before cultured; 0.77±0.063), and CECs in RestM (0.73±0.09), while MitoM showed lower circularities (0.41±0.19). Genes related to collagen type IV and the extracellular matrix, along with the adult CEC markers ATP1A1, ATP1B1, COL8A2, GPC4, and TJP1, were highly expressed in RestM. Conversely, the IL-6, F3, and ITGB3 genes and the non-adult CEC markers CD44, CNTN3, and CD166 were more expressed in MitoM. Overall, from the transcriptome, we identified 832 differentially expressed probes. A functional analysis of the 308 human annotated differentially expressed genes revealed around 13 functional clusters related to important biological terms, such as extracellular matrix, collagen type 4, immune responses, cell proliferation, and wound healing. Quantitative PCR and immunocytochemistry confirmed the overexpression of ATP1A1, TJP1, and GPC4 in CECs in RestM. Conclusions: The addition of a stabilization step during CEC culture improves the cells' morphology and molecular identity, which agrees with transcriptome data. This suggests that stabilization is useful for studying the plasticity of the corneal endothelium's morphology, and stabilization is proposed as a necessary step in corneal endothelium engineering.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Endotélio Corneano
/
Células Endoteliais
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Mol Vis
Assunto da revista:
BIOLOGIA MOLECULAR
/
OFTALMOLOGIA
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
México
País de publicação:
Estados Unidos