Functional analysis of serially expanded human iPS cell-derived RPE cultures.
Invest Ophthalmol Vis Sci
; 54(10): 6767-78, 2013 Oct 17.
Article
em En
| MEDLINE
| ID: mdl-24030465
ABSTRACT
PURPOSE:
To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures.METHODS:
Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE).RESULTS:
RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels.CONCLUSIONS:
Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Células-Tronco Embrionárias
/
Epitélio Pigmentado da Retina
Tipo de estudo:
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Invest Ophthalmol Vis Sci
Ano de publicação:
2013
Tipo de documento:
Article